Treatment of rhinosinusitis with P-glycoprotein inhibitors

ABSTRACT

Provided herein are, inter alia, methods for treating rhinosinusitis with P-glycoprotein inhibitors. A subject having rhinosinusitis is identified and then treated by administration to the subject an effective amount of a P-gp inhibitor. The subject having rhinosinusitis can be identified by one of skill in the art based on known methods, e.g., based on detection of the presence of symptoms, by endoscopy, or by computed tomography. The efficacy of the treatment can be monitored by methods known in the art, e.g., by monitoring symptoms, by endoscopy or computed tomography. The P-glycoprotein inhibitor can be delivered to the subject&#39;s nasal passage and sinuses by an inhalation device, by flushing, by spraying, or by an eluting implant surgically placed in the subject&#39;s nasal passage or sinuses. The P-glycoprotein inhibitor can also be administered in combination with one or both of a corticosteroid and an antibiotic.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.14/655,662, filed on Jun. 25, 2015, which is a 371 U.S. NationalApplication of PCT/US2013/077945, filed on Dec. 27, 2013, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 61/746,290,filed on Dec. 27, 2012, the entire contents of which are herebyincorporated by reference.

TECHNICAL FIELD

This invention relates to treatment of rhinosinusitis in a subject, andmore particularly to methods for treating rhinosinusitis in a subjectwith P-glycoprotein inhibitors.

BACKGROUND

Paranasal sinuses are four pairs of air-filled cavities connecting tothe nasal passage. The paranasal sinuses are named after the cranialbones in which they are located: the frontal sinuses, the maxillarysinuses, the ethmoid sinuses, and the sphenoid sinuses. A membranelining the paranasal sinuses secretes mucus, which drains into the nasalpassage through a small channel in each sinus. Healthy sinuses aresterile and contain no bacteria. In contrast, the nasal passage normallycontains many bacteria that enter through the nostrils as a personbreathes.

A number of factors and processes are involved in maintaining healthysinuses. The mucus secreted by the membrane lining must be fluid butsticky, in order to flow freely yet absorb pollutants and entrapbacteria. It must also contain sufficient amounts of bacteria-fightingsubstances such as antibodies. Additionally, small hair-like projectionscalled cilia, located in the nostril, must beat in unison to propelmucus outward, in order to expel bacteria and other particles. Moreover,the mucous membranes themselves must be intact, and the sinus passagesmust be open to allow drainage and the circulation of air through thenasal passage. When one or more of these processes or factors are amiss,causing obstruction of the sinus passage, an infection called sinusitisdevelops.

Sinusitis is an inflammation of the mucous membrane lining one or moreparanasal sinuses. Rhinitis is an inflammation of the mucous membranelining the nasal passage. Rhinitis and sinusitis usually coexist and areconcurrent in most individuals; thus most guidelines and experts nowhave adopted the term rhinosinusitis (Fokkens et al., Rhinology 2012March; 50 (Suppl 23): S5).

The symptoms of rhinosinusitis include nasal congestion and obstruction,colored nasal discharge, anterior or posterior nasal drip. Subjects mayalso experience facial pain or pressure, and in severe cases, suffer areduction or a loss of smell (Fokkens et al., 2012). There are twodifferent types of rhinosinusitis: acute and chronic. Acuterhinosinusitis is characterized as rhinosinusitis with completeresolution of symptoms within 12 weeks, while chronic rhinosinusitislasts longer than 12 weeks, and usually involves tissue damage (Fokkenset al., 2012). Nasal polyps are frequently present in some subjects withchronic rhinosinusitis based on epidemiologic studies.

SUMMARY

Disclosed herein are, inter alia, methods for treating rhinosinusitiswith P-glycoprotein inhibitors.

In some embodiments, a subject having rhinosinusitis is identified andtreated by administration to the subject an effective amount of a P-gpinhibitor. The subject having rhinosinusitis may be identified by one ofskill in the art based on known methods, e.g., based on detection of thepresence of symptoms, by endoscopy, or by computed tomography. Theefficacy of the treatment may be monitored by methods known in the art,e.g., by monitoring symptoms, by endoscopy or computed tomography.

In one aspect, a subject with rhinosinusitis is treated with a P-gpinhibitor in an amount sufficient to inhibit P-gp expression and/oractivity. The P-gp inhibitor could be a first generation compound, e.g.verapamil, cyclosporin A, anti-hypertensive, reserpine, quinidine oryohimbine, tamoxifen, or toremifena. Preferably, the P-gp inhibitor is asecond or third generation compound, e.g. PSC 833, R-verapamil, VX-710,GF120918, MS-209, R101933, LY335979, OC144093, XR9051, or XR9576.

In another aspect, a subject with rhinosinusitis is treated with a P-gpinhibitor in an amount sufficient to decrease P-gp expression in thesubject's sinonasal epithelial cells, either transcriptionally orposttranscriptionally.

In some embodiments, the P-gp inhibitor is administered systemically. Inother embodiments, the P-gp inhibitor is administered locally to thesubject's nasal passage and sinuses by an inhalation device, byflushing, spraying, irrigation, nebulization, atomization, or a drugeluting vehicle.

In some embodiments, a subject with rhinosinusitis is treated with aP-gp inhibitor in combination with other conventional treatments, e.g.,drugs such as corticosteroids and/or antibiotics, to potentiate theeffect of treatment.

In some embodiments, when a subject with rhinosinusitis has nasalpolyps, surgical removal of such nasal polyps can be performed inaddition to administration of a P-gp inhibitor to the subject. Thus, asubject with rhinosinusitis may undergo both surgery and treatment witha P-gp inhibitor.

In some embodiments, a subject with rhinosinusitis has eosinophilicsinusitis and/or other forms of mucosal inflammation.

In some embodiments, a subject continues to experience symptoms ofchronic sinusitis after a sinus surgery, and a P-gp inhibitor-elutingimplant, stent, or spacer is used to maintain sinus patency in thesubject. The P-gp inhibitor eluting device can be made frombioabsorbable material so that the implant will be absorbed within ashort period of time after the implantation and no surgical removal ofthe implant is necessary. The P-gp inhibitor eluting device can be inthe form of solid, semisolid, gel, polymer, or particle.

In some embodiments, the P-glycoprotein inhibitor is administered incombination with one of both of a corticosteroid and/or an antibiotic.The corticosteroid can be, e.g., selected from dexamethasone,prednisone, prednisolone, triamcinolone, cortisol, budesonide,mometasone, fluticasone, flunisolide, and betamethasone. The antibioticcan be, e.g., selected from erythromycin, doxycycline, tetracycline,penicillin, beta-lactam, macrolide, fluoroquinolone, cephalosporin, andsulfonamide.

In some embodiments, a kit for treating rhinosinusitis in a subject isprovided. Such a kit comprises a pharmaceutical composition comprisingan effective amount of a P-gp inhibitor, and a device for delivering thepharmaceutical composition to the subject's nasal passage and sinuses.The device may deliver the pharmaceutical composition to the subject'snasal passage and sinuses in a liquid or an aerosolized form. In someembodiments, the kit also includes a corticosteroid and/or anantibiotic, in the same pharmaceutical composition as the P-gp inhibitoror in a separate composition.

As used herein, “treatment” means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. As used herein, amelioration of the symptoms of aparticular disorder refers to any lessening, whether permanent ortemporary, lasting or transient that can be attributed to or associatedwith treatment by the compositions and methods of the presentdisclosure.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutic amount is one that achievesthe desired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a therapeutic compound (i.e., aneffective dosage) depends on the therapeutic compounds selected. Thecompositions can be administered from one or more times per day to oneor more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the therapeutic compounds described herein caninclude a single treatment or a series of treatments.

The term “subject” is used throughout the specification to describe ananimal, human or non-human, to whom treatment according to the methodsof the present invention is provided. Veterinary and non-veterinaryapplications are contemplated. The term includes, but is not limited to,mammals, e.g., humans, other primates, pigs, rodents such as mice andrats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheepand goats. Typical subjects include humans, farm animals, and domesticpets such as cats and dogs.

The term “rhinosinusitis” as used herein includes acute and chronicrhinosinusitis, either with or without the presence of nasal polyps.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph showing Q-FIHC ratios of P-gp staining intensityat two tissue subsites (sinus and septum) among the control subjectgroups (Control), the subject group having chronic rhinosinusitis butwithout nasal polyps (CRS), and the subject group having chronicrhinosinusitis with nasal polyps (CRSwNP) (* p<0.001, ** p=0.002).Dashed line denotes a ratio of 1 suggesting no increased epithelialstaining over background.

FIGS. 2A-C are a set of matched photomicrograph images (bar=100 μm) ofsinus tissue representing P-gp immunostaining (top panel), propidiumiodide nuclear stain (middle panel), and H&E staining (lower panel) inCRSwNP (A), CRS (B), and Control (C) subjects. Note the increased P-gpepithelial staining in the CRSwNP group relative to CRS and control.Inset images in the top row represent negative control slides.

FIGS. 3A-B are a set of high magnification histologic images (bar=50 μm)of P-gp immunostaining in CRSwNP (A) and Control (B) subjects withmatched propidium iodide nuclear stains (lower panel). Note thecircumferential membranous P-gp expression subtending both the apicaland basolateral surfaces of the epithelial cells.

FIG. 4 is a graph showing Q-FIHC staining intensity of membranous P-gpin PNECCs exposed to culture media alone (BEGM) versus BEGM with 0.05mg/mL of LPS for 23 hours (h). Corrected luminosity refers to the totalluminosity divided by the total cell area per field. Exposure to LPSresulted in a significant increase in staining intensity.

FIG. 5 is a set of fluorescent images in primary human sinonasalepithelial cell culture depicting P-gp staining (upper windows)following exposure to BEGM or BEGM+LPS. The lower windows depict thematched propidium iodide (PI) nuclear staining for cellularlocalization. P-gp staining intensity was quantified in FIG. 4.

FIG. 6 is a box graph showing membranous P-gp expression determined byELISA following membrane protein extraction. The significant increase inexpression following LPS exposure was again seen confirming the findingsby IHC. There was no significant difference in expression seen betweencells exposed to LPS and those exposed to LPS along with a P-gpinhibitor (PSC 833, p=0.115).

FIG. 7 is a bar graph of rhodamine 123 accumulation assay demonstratingconcentration of intracellular rhodamine as compared to baselinefollowing inhibition of P-gp using either PSC 833 or verapamil. Theevident dose response using two separate inhibitors confirmed that theP-gp expressed in PNECCs is sensitive to inhibition. The accumulation inrhodamine 123 over baseline seen with 8micM of PSC 833 suggested thatthe alterations in cytokine secretion seen following PSC 833 exposure atthis concentration may be directly attributable to P-gp inhibition.

FIG. 8 is a box graph showing secreted IL-6 concentration (normalized tototal media protein) in the control condition (CTRL) following exposureto culture media alone as compared to PNECCs exposed to media+LPS (0.05mg/mL), and media+LPS (0.05 mg/mL)+PSC 833 (8micM). Note the significantreduction in LPS stimulated IL-6 secretion following inhibition of P-gpwith PSC 833.

FIG. 9 is a box graph showing secreted GM-CSF concentration (normalizedto total media protein) in the control condition (CTRL) followingexposure to culture media alone as compared to PNECCs exposed tomedia+LPS (0.05 mg/mL), and media+LPS (0.05 mg/mL)+PSC 833 (8micM). Notethe significant reduction in LPS stimulated GM-CSF secretion followinginhibition of P-gp with PSC 833.

FIG. 10 is a box graph showing secreted TSLP concentration (normalizedto total media protein) in the control condition (CTRL) followingexposure to culture media alone as compared to PNECCs exposed tomedia+LPS (0.05 mg/mL), and media+LPS (0.05 mg/mL)+PSC 833 (8micM). Notethe significant reduction in LPS stimulated TSLP secretion followinginhibition of P-gp with PSC 833.

FIG. 11 is a box graph showing secreted IL-8 concentration (normalizedto total media protein) in the control condition (CTRL) followingexposure to culture media alone as compared to PNECCs exposed tomedia+LPS (0.05 mg/mL), and media+LPS (0.05 mg/mL)+PSC 833 (8micM). Inthis case PSC 833 failed to significantly impair IL-8 secretionsuggesting that P-gp mediated secretion is selective to specificcytokines.

FIG. 12 is a bar graph showing that treatment with the P-gp inhibitorPSC 833 resulted in increased intracellular dexamethasone retention inprimary sinonasal epithelial cells relative to uninhibited cells.

FIG. 13A is a bar graph showing that treatment with the P-gp inhibitorVerapamil or Zosuquidar resulted in increased intracellular prednisoneretention in primary sinonasal epithelial cells relative to uninhibitedcells. FIG. 13B is a bar graph showing that treatment with the P-gpinhibitor Verapamil or Zosuquidar resulted in increased intracellulardexamethasone retention in primary sinonasal epithelial cells relativeto uninhibited cells.

FIG. 14 is a line graph showing that treatment with the P-gp inhibitorPSC 833 resulted in increased intracellular dexamethasone retention innasal polyp explants relative to uninhibited polyp explants.

FIG. 15 is a box graph showing that GM-CSF secretion is reduced inLPS-stimulated cells treated with both dexamethasone and PSC833 ascompared to dexamethasone treatment alone.

FIG. 16A is a set of fluorescent images of epithelial calcein stainingin nasal polyp explants with or without the presence of PSC 833(bar=50μm). FIG. 16B is a bar graph demonstrating the corrected luminosity ofboth septal and nasal polyps explants with or without the presence ofPSC 833(n=4, each). The increase in calcein luminosity following PSC 833mediated inhibition is proportionate to the degree of P-gp activity.

FIG. 17A is a FIHC image demonstrating pattern of P-gp expression insubmerged CRSwNP HSNECC with propidium iodide (PI) nuclear counterstain.Inset represents control slide with primary antibody omitted. FIG. 17Bis a box and whisker plot of membranous P-gp expression (ng/mL)normalized to total cytoplasmic protein (mcg/mL) demonstrating asignificant upregulation following LPS exposure as compared to cellsexposed to media alone.

FIG. 18 is a dot plot demonstrating a significant dose-dependentinhibition of P-glycoprotein following PSC 833 exposure. The increase inmean calcein fluorescence following exposure to varying concentrationsof PSC 833 relative to uninhibited control cells is proportionate to asuccessive reduction in P-gp activity.

FIGS. 19A and 19B are box and whisker plots of cytokine secretion ofGM-CSF (A) and IL-6 (B) under control, LPS stimulated, and LPSstimulated+P-gp inhibitor conditions (n=5, each). The y-axis representssecreted cytokine concentration (pcg/mL) normalized to total mediaprotein (mcg/mL)×100. FIGS. 19C and 19D are scatter plots showing thepositive correlation between LPS stimulated normalized GM-CSF (C) andIL-6 (D) secretion and membranous P-gp expression.

FIGS. 20A and 20B are box and whisker plots of cytokine secretion ofIL-8 and IL-25 under control, LPS stimulated, and LPS stimulated+P-gpinhibitor conditions. The y-axis represents secreted cytokineconcentration (pcg/mL) normalized to total media protein (mcg/mL)×100.FIGS. 20C and 20D are scatter plots showing the lack of correlationbetween LPS stimulated normalized IL-8 (C) and IL-25 (D) secretion andmembranous P-gp expression.

FIG. 21A is a bar graph demonstrating the mean P-gpepithelial/background staining ratios between the low and high P-gpexpressing patient groups. FIG. 21B is a box and whisker plotdemonstrating the distribution of eosinophils/hpf between the low andhigh P-gp expressing patient groups. FIG. 21C is a box and whisker plotdemonstrating the distribution of Lund-Mackay scores between the low andhigh P-gp expressing patient groups.

FIGS. 22A and B are fluorescent immunohistochemical images of mucosadepicting representative (A) low epithelial P-gp expression and (B) highepithelial P-gp expression (bar=50 μM, lower right inset representsnegative control in which the primary antibody was omitted). FIGS. 22Cand D are matched high powered (400×) H&E stromal images depicting theabsence (C) and presence (D) of mucosal eosinophilia (black arrowsdenote individual eosinophils). Note the thickened basement membrane inC is consistent with CRSsNP. FIGS. 22E and F are coronal CT scansdemonstrating increased radiographic inflammation in the patient withhigh P-gp expression (F) relative to the patient with low P-gpexpression (E).

FIG. 23A is a bar graph demonstrating the mean P-gpepithelial/background staining ratios between the low and highP-gp-expressing patient groups. FIG. 23B is a box and whisker plotdemonstrating the distribution of serum eosinophil between the low andhigh P-gp-expressing patient groups.

FIG. 24A is a box and whisker plot demonstrating the distribution of GOSbetween the low and high P-gp-expressing patient groups. FIG. 24B is abox and whisker plot demonstrating the distribution of KOS between thelow and high P-gp expressing patient groups. FIG. 24C is a scatter plotof both osteitis scoring systems demonstrating a significant, highcorrelation between KOS and GOS.

DETAILED DESCRIPTION

About 16% of the adult population in the United States suffers chronicrhinosinusitis (Blackwell et al., Vital Health Stat. 10. 2002 May;(205):1-109). According to U.S. Centers for Disease Control andPrevention, over 30 million Americans have sinusitis, resulting in about200,000 to 500,000 sinus surgeries and 23 million missed work days peryear.

The primary objectives for treating rhinosinusitis are reduction ofinflammation, eradication of infection, draining of the sinuses, andensuring that the paranasal sinuses are and remain open.

Described herein are methods for treating rhinosinusitis in subjectswith P-glycoprotein (P-gp) inhibitors. P-glycoprotein (P-gp) is an ATPdependent efflux pump. The data shown herein demonstrate that P-gp isoverexpressed in subjects with rhinosinusitis, and P-gp functions as animmunomodulator through regulation of epithelial cytokine secretion.Therefore, P-gp contributes to the etiopathogenesis of sinonasalinflammation, and P-gp inhibitors can be used for treating subjects withrhinosinusitis. P-gp inhibitors can also be used in combination withother conventional treatments, e.g., drugs such as corticosteroidsand/or antibiotics, to potentiate the effect of treatment by enhancingintracellular retention. For example, P-gp inhibitors can be used incombination with a corticosteroid, e.g., selected from dexamethasone,prednisolone, triamcinolone, cortisol, prednisone, budesonide,mometasone, fluticasone, flunisolide, and betamethasone. In someembodiments, P-gp inhibitors are used in combination with an antibiotic,e.g., selected from macrolides, e.g., erythromycin; penicillins, e.g.,amoxicillin, beta-lactam, ampicillin; tetracyclines, e.g., doxycycline,tetracycline; sulfonamides, e.g. mafenide, sulfacetamide;fluoroquinolones; and cephalosporins, e.g., ceftaroline fosamil,ceftobiprole. In some embodiments, P-gp inhibitors are used incombination with a corticosteroid and an antibiotic.

Conventional Treatment of Rhinosinusitis

The present methods can be used in combination with present conventionaltreatments of rhinosinusitis, e.g., as follows. Most subjects withrhinosinusitis caused by bacteria are treated with antibiotics alongwith a nasal decongestant. The most common side effect for nearly allantibiotics is gastrointestinal distress. Certain drugs, including someover-the-counter medications, interact with antibiotics, and allantibiotics carry the risk for allergic reactions, which can be seriousin some cases. Failure to take all prescribed antibiotics may increasethe risk for reinfection and also for development ofantibiotic-resistant bacteria. The usefulness of antibiotics in treatingchronic sinusitis is highly debated, as some symptoms persist even afterprolonged courses of antibiotics. Furthermore, a vast majority ofsinusitis is caused by viruses and will not respond to antibiotictreatment.

Nasal decongestants may dry out the affected areas and damage tissues.With prolonged use, nasal decongestants become ineffective, and thetendency is to increase the frequency of use. Withdrawal fromover-frequent decongestant use can itself cause symptoms ofrhinosinusitis and the return of nasal congestion, a phenomenon known asthe “rebound effect.” Short-acting nasal decongestants may cause reboundeffect after only eight hours. Eventually, the inflammation can becomeworse than before the decongestant was taken. Thus, nasal decongestantsare generally recommended for no more than one to three days of usebecause of this risk.

Steroid nasal sprays are commonly used to treat inflammation in chronicsinusitis. For subjects with severe chronic sinusitis, doctors mayprescribe oral steroids, such as prednisone. Since oral steroids haveserious side effects, they are prescribed only when other medicationshave not been effective.

When medications fail, surgery may be the only alternative in treatingchronic sinusitis. Presently, the most commonly done surgery isfunctional endoscopic sinus surgery, in which the sinuses are reachedthrough the nasal passage via endoscopy, and the diseased and thickenedtissues from the sinuses are removed to enlarge the sinus passageway tothe nostril and allow for drainage and improved topical drug delivery.This type of surgery is less invasive than traditional open sinussurgery techniques. Symptoms of chronic sinusitis sometimes persistafter surgery, however, because of continued inflammation, growth of newnasal polyps, or scarring from the procedure.

P-Glycoprotein

P-glycoprotein is a 170-kDa glycoprotein encoded by the MDR1 (ABCB1)gene located on chromosome 7q21.12 and was first identified in the CHOcell line (Fernandez et al., J Pharm. Pharm. Sci. 2004 Nov. 17;7(3):359-71). P-gp is a member of the ATP-binding cassette (ABC)transporter family and is capable of energy dependent transport of avariety of intracellular substrates (Golden et al., J Pharm Sci. 2003;92(9):1739-53). P-gp is located within the plasma membrane and functionsto extrude xenobiotic agents against their concentration gradient(Ehrhardt et al., Pharm. Res. 2003 April; 20(4):545-51). Substraterecognition of P-gp occurs by a variety of mechanisms including thepresence of electron donor groups which bind putative reactive hydrogenbonding sites in the interior channels formed by the 12 transmembranehelices (Golden et al., 2003).

P-gp is constitutively expressed on multiple cell types including theapical membrane of intestinal mucosal cells, the brush border of renalproximal tubules, the blood-brain barrier, and lower airway epithelialcells (Bleier B S, Int. Forum Allergy Rhinol. 2012; 2:122-125). Due tothe selective distribution at the port of drug entry and exit, P-gpfunctions as a biochemical barrier for entry of xenobiotics and as avacuum cleaner to expel them from the organs, such as brain, liver,kidney, and ultimately from systemic circulation (Varma et al.,Pharmacological Research 2003; 48: 347-359). This xenobiotic excretionfunction belies the role of P-gp in reducing the systemicbioavailability of a variety of drugs. Through increased expression andactive drug efflux in malignancy, P-gp has also been shown to conferchemotherapeutic resistance (Fernandez et al., 2004).

While the efflux behavior of P-gp is well established, the potential forthe role of P-gp as an immunomodulator is a new concept. In a P-gpknockout mouse (Mdr1a/1b^(−/−)), there was diminished dendritic cell(DC) maturation and subsequent DC induced T-cell response whichcorrelated with decreased Th1 and Th2 cytokine levels (Kooij et al.,PLoS One. 2009 Dec. 8; 4(12):e8212). In the lower airway, inhalationalsteroid exposure has been shown to decrease the expression of lymphocyteP-gp (Kopriva et al., J Asthma. 2009 May; 46(4):366-70).

P-Glycoprotein Inhibitors

A number of inhibitors of P-gp are known in the art (Varma et al.,2003). In general, P-gp can be inhibited (1) by blocking its substratebinding site; (2) by interfering with its ATPase activity (Shapiro, etal., Biochem Pharmacol 1997; 53:587-96); or (3) by decreasing itsexpression level either transcriptionally or posttranscriptionally.(Drori et al., Eur J Biochem 1995; 228:1020-9).

Based on specificity and affinity, P-gp inhibitors are classified intothree generations. First-generation P-gp inhibitors are knownpharmacological compounds that are in clinical use, or were developedfor, for other indications but have been shown to inhibit P-gp. Theseinclude calcium channel blockers such as verapamil; immunosuppressantslike cyclosporin A; anti-hypertensives, reserpine, quinidine andyohimbine; and anti-estrogens like tamoxifen and toremifena (Varma etal., 2003). The usage of these compounds has been limited by theirtoxicity due to the high serum concentrations achieved with the dosethat is required to inhibit P-gp when administered systemically.

Second-generation P-gp modulators are agents that lack thepharmacological activity of the first-generation compounds and usuallypossess a higher P-gp affinity. Second-generation P-gp inhibitorsinclude the non-immunosuppresive analogues of cyclosporin A such as PSC833 (Valspodar: 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoicacid]-7-L-valine-cyclosporin A); verapamil isomers such as D-isomer ofverapamil, R-verapamil, and dexverapamil; and other inhibitors such asVX-710 (Biricodar: 1,7-di(pyridin-3-yl)heptan-4-yl(2S)-1-[oxo(3,4,5-trimethoxyphenyl)acetyl]piperidine-2-carboxylate);GF120918 (Elacridar:N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-5-methoxy-9-oxo-9,10-dihydroacridine-4-carboxamidehydrochloride); and MS-209 (Dofequidar fumarate:1-(4-(2-hydroxy-3-(quinolin-5-yloxy)propyl)piperazin-1-yl)-2,2-diphenylethanone)(Varma et al., 2003). However, this class of compounds often inhibitstwo or more ABC transporters, leading to some drug-drug interactions.

The third-generation P-gp blockers are under development with theprimary purpose to improve the treatment of multidrug resistant tumorsand to inhibit P-gp with high specificity and toxicity. Examples of thethird-generation P-gp inhibitors include LY335979 (Zosuquidar:(2R)-1-{4-[(1aR,6r,10bS)-1,1-Difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]piperazin-1-yl}-3-(quinolin-5-yloxy)propan-2-ol,trihydrochloride);OC144093(4-[2-[4-[(E)-3-ethoxyprop-1-enyl]phenyl]-4-[4-(propan-2-ylamino)phenyl]-1H-imidazol-5-yl]-N-propan-2-ylaniline);R-101933 (Laniquidar: methyl11-(1-(4-(quinolin-2-ylmethoxy)phenethyl)piperidin-4-ylidene)-6,11-dihydro-5H-benzo[d]imidazo[1,2-a]azepine-3-carboxylate);XR9576 (Tariquidar:N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide);XR9051(3-((Z)—((Z)-5-benzylidene-4-methyl-3,6-dioxopiperazin-2-ylidene)methyl)-N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)benzamide).Some third-generation P-gp modulators such as LY335979, OC144093, andXR9576 are shown to be highly potent and selective inhibitors of P-gpwith a potency of about 10-fold more than the first andsecond-generation inhibitors. (Varma et al., 2003).

Treatment of Rhinosinusitis Using P-Glycoprotein Inhibitors

While the expression of P-gp has been studied in the lower airway, verylittle is known with respect to its presence in the upper airway or itsrelationship to chronic sinonasal inflammation. Furthermore, there is noreport on the expression of P-gp across disease states or between sinusand adjacent intranasal sub sites. Therefore, the pattern and degree ofepithelial P-gp expression was examined in subjects having chronicsinusitis (CRS) with or without nasal polyposis (NP) and in controlsubjects.

The data presented herein show that the expression of P-gp is negligiblein healthy sinus mucosa, but is significantly elevated in the epitheliallayer of sinus mucosa in subjects having CRS with or without NP relativeto other non-diseased sinonasal subsites.

The sinonasal epithelium functions as a barrier organ against theexternal environment and is endowed with an array of innate and adaptiveimmunologic mechanisms to combat extrinsic pathogens. It has beensuggested that sinonasal epithelial cells may function as primary actorsin the initiation and maintenance of chronic sinonasal inflammationthrough the elaboration of an array of cytokines and subsequentrecruitment of professional immune cells (Reh et al., Am. J Rhinol.Allergy. 2010; 24(2):105-9). While these studies suggest that epithelialcells are capable of orchestrating an innate immune response, thepost-translational mechanisms governing non-canonical cytokine secretionat the cellular level are not fully understood.

The data presented herein show that membrane bound P-gp is present andfunctionally active in primary nasal epithelial cells, and P-gpinhibitor (e.g., PSC 833) exposure results in a significant reduction instimulated cytokine secretion of those primary nasal epithelial cells.As demonstrated herein, P-gp participates in modulating cytokinesecretion and inflammation at the nasal mucosal surface. Furthermore,P-gp inhibition in epithelial cells is shown to result in increasedintracellular steroid retention and potentiate the anti-inflammatoryeffect of steroid.

Collectively, the present data show that P-gp is overexpressed in sinusmucosa of subjects having rhinosinusitis, and P-gp functions as animmunomodulator through regulation of epithelial cytokine secretion.Therefore, P-gp contributes to the etiopathogenesis of sinonasalinflammation and P-gp inhibitors are promising novel medicines fortreating rhinosinusitis in order to reduce the cytokine secretion whichleads to the development of sinonasal inflammation.

In some embodiments, a subject having rhinosinusitis is identified andtreated by administering to the subject an effective amount of a P-gpinhibitor. The subject having rhinosinusitis may be identified by one ofskill in the art based on known methods, e.g., based on detection of thepresence of symptoms, by endoscopy, or by computed tomography. Theefficacy of the treatment may be monitored by methods known in the art,e.g., by monitoring symptoms, by endoscopy or computed tomography.Improvements of the subject include a better symptom score, e.g. abetter SNOT-22 or VAS score; a reduction in inflammation or nasal polypburden as revealed by endoscopy, e.g. a better Lund-Kennedy score; or areduction in mucosal thickening or sinus opacification as revealed bycomputed tomography (CT), e.g. a better Lund-Mackay score. The 22-itemSinonasal Outcomes Test (SNOT-22) is a questionnaire encompassing 22major symptoms on rhinosinusitis and nasal polyps, and serves as avaluable tool to measure the severity of a subject's symptoms and theirimpact on health-related quality of life (Quintanilla-Dieck, et al.,International Forum of Allergy & Rhinology 2012; 2(6):437-443). TheSNOT-22 assessed 12 nasal- and sinus-related symptoms (nasal blockage,loss of sense of taste and smell; need to blow nose, sneezing, runnynose, cough, postnasal discharge, thick nasal discharge, ear fullness,dizziness, ear pain, and facial pain/pressure) and 10 psychological andbehavioral symptoms (difficulty falling asleep, waking up at night, lackof a good night's sleep, waking up tired, fatigue, reduced productivity,reduced concentration, frustrated/restless/irritable, sad, andembarrassed) with participants scoring each symptom on a scale of 0(absent) to 5 (severe) on average for the last week, for a total scorerange of 0 to 100. The SNOT-22 score is the mean for the 22 scores(Piccirillo et al., Otolaryngol Head Neck Surg 2002; 126:41-47). The10-symptom visual analog (VAS) scale is a questionnaire based on themajor and minor symptom diagnostic criteria for CRS as described by theAmerican Academy of Otolaryngology—Head and Neck Surgery TFR. The VASassessed subject-reported severity of each of the following symptoms onaverage experienced during the prior week: nasal drainage of pus, nasalobstruction/congestion, impaired sense of smell, facial pressure/pain,headache, bad breath, weakness/fatigue, dental pain, ear fullness/pain,and cough (Ryan, et al., Laryngoscope 2011; 121:674-678). TheLund-Kennedy endoscopy scoring system quantifies the pathologic statesof the nose and paranasal sinuses as assessed by nasal endoscopy,focusing on the presence of polyps, discharge, edema, scarring oradhesions, and crusting (Ryan, et al., 2011). The Lund Mackay CT scoringsystem is the most widely used CT grading system for chronicrhinosinusitis. This scoring system consists of a scale of 0-2 dependenton the absence (0), partial (1) or complete (2) opacification of thesinus system and the osteomeatal complex as assessed by CT imaging(Hopkins et al., Otolaryngology—Head and Neck Surgery 2007;137:555-561).

In some embodiments, a subject with rhinosinusitis is treated with aP-gp inhibitor in an amount sufficient to inhibit P-gp function. TheP-gp inhibitor could be a first generation compound, e.g. a calciumchannel blocker such as verapamil, an immunosuppressant like cyclosporinA, an anti-hypertensive, a reserpine, a quinidine or yohimbine, or ananti-estrogen like tamoxifen or toremifena. Preferably, the P-gpinhibitor is a second or third generation compound, e.g. PSC 833,D-isomer of verapamil, dexverapamil, VX-710, GF120918, MS-209, R101933,LY335979, OC144093, XR9051, or XR9576.

In other embodiments, a subject with rhinosinusitis is treated with aP-gp inhibitor in an amount sufficient to decrease P-gp expression inthe subject's sinonasal epithelial cells, either transcriptionally orposttranscriptionally.

In some embodiments, the P-gp inhibitor is administered systemically,e.g., orally, intravenously, intradermally, or subcutaneously. In otherembodiments, the P-gp inhibitor is administered locally to the subject'snasal passage and sinuses by an inhalation device, by flushing, or byspraying. In some embodiments, a subject with rhinosinusitis is treatedwith nasal drops or sprays comprising an effective amount of a P-gpinhibitor. An effective amount of the P-gp inhibitor can be delivered tothe subject's nasal passage and sinuses in a liquid form by flushing orspraying. An effective amount of a P-gp inhibitor can also be deliveredto the nasal passage and sinuses of a subject with rhinosinusitis in anaerosolized form by an inhalation device, such as a nebulizer, aninhaler, or an OptiNose.

In some embodiments, a subject with rhinosinusitis is treated with aP-gp inhibitor in combination with other conventional treatments, e.g.,drugs such as corticosteroids and/or antibiotics, to potentiate theeffect of treatment. For example, P-gp inhibitors may be used incombination with a corticosteroid selected from dexamethasone,prednisolone, triamcinolone, cortisol, prednisone, budesonide,mometasone, fluticasone, flunisolide, and betamethasone. In someembodiments, P-gp inhibitors are used in combination with an antibioticselected from macrolides, e.g., erythromycin; penicillins, e.g.,amoxicillin, beta-lactam, ampicillin; tetracyclines, e.g., doxycycline,tetracycline; sulfonamides, e.g. mafenide, sulfacetamide;fluoroquinolones; and cephalosporins, e.g., ceftaroline fosamil,ceftobiprole. In some embodiments, P-gp inhibitors are used incombination with a corticosteroid and an antibiotic.

In some embodiments, when a subject with rhinosinusitis has nasalpolyps, surgical removal of such nasal polyps can be performed inaddition to administration of a P-gp inhibitor to the subject. Thus, asubject with rhinosinusitis may undergo both surgery and treatment witha P-gp inhibitor.

In some embodiments, a subject continues to experience symptoms ofchronic sinusitis after a sinus surgery, and a P-gp inhibitor-elutingimplant, stent, or spacer is used to maintain sinus patency in thesubject. During the sinus surgery, a P-gp inhibitor eluting device isimplanted, e.g., in the ostia of the paranasal sinuses to prop open theostia while locally eluting a P-gp inhibitor to reduce inflammation ofthe sinonasal epithelium after the surgery. The P-gp inhibitor elutingdevice can be made from bioabsorbable material so that the implant willbe absorbed within a short period of time after the implantation and nosurgical removal of the implant is necessary. The P-gp inhibitor elutingdevice can be in the form of solid, semisolid, gel, polymer, orparticle. In some embodiments, the P-gp inhibitor eluting device is abioabsorbable gel such as an alginate gel (e.g., sodium alginate), acellulose-based gel (e.g., carboxymethyl cellulose or carboxyethylcellulose), or a chitosan-based gel (e.g., chitosan glycerophosphate;see, e.g., Bleier et al., Am J Rhinol Allergy 23, 76-79, 2009).

In some embodiments, a tissue sample, e.g., a sinus mucosal biopsysample, can be obtained from a subject having rhinosinusitis and one ormore tests can be performed on these biopsy samples to assist inselecting a therapy for the subject. For example, levels of P-gpexpression in the nasal epithelium can be determined using methods knownin the art, e.g., quantitative fluorescent immunohistochemistry. When aP-gp expression level in the nasal epithelium is determined to be abovea threshold (i.e., a reference level), a therapy comprising a P-gpinhibitor as described herein can be selected to treat rhinosinusitis inthe subject.

In some embodiments, sinus mucosal biopsy samples from a subject havingrhinosinusitis and the average number of eosinophils per high poweredfield can be calculated, e.g., using light microscopy and staining withhematoxylin and eosin. As demonstrated herein, P-gp expression levelscorrelate with tissue eosinophilia, thus high levels of tissueeosinpophelia (i.e., levels above a reference level) can be used as aproxy for high levels of P-Gp expression. A therapy as described hereincomprising administration of a P-gp inhibitor can be selected to treatrhinosinusitis in the subject when the average number of eosinophils perhigh powered field is determined to be above a threshold (i.e.,reference level).

In some embodiments, computed tomography (CT) can be performed to scoreosteitis in a subject having rhinosinusitis. For example, a KennedyOsteitis Score (KOS) (Lee J T, Kennedy D W, Palmer J N, Am J Rhinol20:278-282, 2006) or Global Osteitis Score (GOS) (Georgalas C, VidelerW, Freling N, Clin Otolaryngol 35:455-461, 2010) can be determined forthe bony walls of the paranasal sinuses as previously described. Asdemonstrated herein, these osteitis scores correlate with P-gpexpression level in patients having chronic sinusitis, and thus a highosteitis score can be used as a proxy for high P-gp expression levels.When an osteitis score is determined to be above a threshold (i.e., areference level), a therapy as described herein comprisingadministration of a P-gp inhibitor can be selected to treatrhinosinusitis in the subject.

One of skill in the art would readily be able to determine and select asuitable reference level. For example, a reference level can bedetermined as a median, average, or cutoff point for a percentile of thepopulation (e.g., the cutoff for the top half, top tertile, topquartile, top quintile, and so on). A reference level can be selectedthat represents a level of P-gp expression, eosinophilia, KOS, or GOS ina subject that would be likely to benefit from treatment with a P-gpinhibitor, and levels at or above that reference level indicate that thesubject should be treated with a method comprising administration of aP-gp inhibitor as described herein.

Methods for Selecting a Subject for Participation, or StratifyingSubjects, in a Clinical Study

Also provided are methods of selecting a subject for participation in,or stratifying subjects in, a clinical study of a treatment forrhinosinusitis. Such methods can include determining a level ofepithelial P-gp expression in a sinus mucosal biopsy sample from asubject, comparing the P-gp level in the sample to a reference P-gplevel, and selecting for participation a subject having an elevated P-gplevel in the sample compared to the reference P-gp level in a clinicaltrial of a treatment for rhinosinusitis, or stratifying subjects in aclinical trial based on P-gp levels. In some embodiments, a subject canbe excluded from participation in a clinical study of a treatment forrhinosinusitis if the subject has no significant change or a decrease inthe P-gp level in the sample compared to the reference P-gp level.

Also provided are methods of selecting a subject for participation in,or stratifying subjects in, a clinical study for a treatment forrhinosinusitis. Such methods include determining a P-gp level in a firstbiopsy sample obtained from a subject at a first time point, determininga P-gp level in a second biopsy sample obtained from the subject at asecond time point, comparing the P-gp level in the first biopsy sampleto the P-gp level in the second biopsy sample, and selecting a subjecthaving an elevated P-gp level in the second biopsy sample compared tothe P-gp level in the first biopsy sample for participation in aclinical trial of a treatment for rhinosinusitis, or stratifyingsubjects in a clinical trial based on changing P-gp levels. In someembodiments, a subject can be excluded from participation in a clinicalstudy of a treatment for rhinosinusitis if the subject has nosignificant change or a decrease in the P-gp level determined at thesecond time point compared to the P-gp level determined at the firsttime point. In some embodiments, the treatment for rhinosinusitis is apharmacological treatment (e.g., administration of one or morepharmaceutical agents) or the implantation of an eluting implant, stent,or spacer.

In some embodiments, additional clinical scores can be used to assist inselecting a subject for participation in, or stratifying subjects in, aclinical study of a treatment for rhinosinusitis. For example, sinusmucosal biopsy samples can be processed for hematoxylin and eosinstaining and the average number of eosinophils per high powered fieldcan be calculated and used in selecting a subject for participation in,or stratifying subjects in a clinical study of a treatment forrhinosinusitis. For example, subjects with levels of eosinophilia abovea reference level can indicate that the subject should be selected orstratified.

In some embodiments, computed tomography (CT) can be performed to scoreosteitis in a subject having rhinosinusitis. The osteitis score, e.g.,Kennedy Osteitis Score (KOS) or Global Osteitis Score (GOS) can be usedin selecting a subject for participation in, or stratifying subjects in,a clinical study of a treatment for rhinosinusitis. For example,subjects with GOS or KOS above a reference level can indicate that thesubject should be selected or stratified.

The clinical studies may be performed by a health care professional(e.g., a physician, a physician's assistant, a nurse, a phlebotomist, ora laboratory technician) in a health care facility (e.g., a hospital, aclinic, or a research center). The biopsy samples may be obtained fromsubjects that present with one or more (e.g., at least two, three, four,or five) symptoms of rhinosinusitis.

Pharmaceutical Compositions, Dosage, and Methods of Administration

The methods of treatment described herein also include the use ofpharmaceutical compositions, which include P-gp inhibitors describedherein as active ingredients. In some embodiments the composition alsoincludes one or more supplementary active compounds incorporatedtherein, e.g., one or more corticosteroids and/or one or moreantibiotics. The corticosteroid can be, e.g., selected fromdexamethasone, prednisone, prednisolone, triamcinolone, cortisol,budesonide, mometasone, fluticasone, flunisolide, or betamethasone. Theantibiotic can be, e.g., selected from erythromycin, doxycycline,tetracycline, penicillin, beta-lactam, macrolide, fluoroquinolone,cephalosporin, and sulfonamide. Also included are the pharmaceuticalcompositions themselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, NY). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders, for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration of a therapeutic compound as described hereincan also be by transmucosal or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished through the use of nasalsprays or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art.

In one embodiment, the therapeutic compounds are prepared with carriersthat will protect the therapeutic compounds against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

In some embodiments, a kit for treating rhinosinusitis in a subject isprovided. Such a kit comprises a pharmaceutical composition comprisingan effective amount of a P-glycoprotein inhibitor, optionally acorticosteroid and/or an antibiotic, and a device for delivering thepharmaceutical composition to the subject's nasal passage and sinuses,such as a nebulizer, an inhaler, or an OptiNose. The device may deliverthe pharmaceutical composition to the subject's nasal passage andsinuses in a liquid or an aerosolized form.

In non-limiting examples, the pharmaceutical composition containing atleast one pharmaceutical agent is formulated as a liquid (e.g., athermosetting liquid), as a component of a solid (e.g., a powder or abiodegradable biocompatible polymer (e.g., a cationic biodegradablebiocompatible polymer)), or as a component of a gel (e.g., abiodegradable biocompatible polymer). In some embodiments, the at leastcomposition containing at least one pharmaceutical agent is formulatedas a gel selected from the group of an alginate gel (e.g., sodiumalginate), a cellulose-based gel (e.g., carboxymethyl cellulose orcarboxyethyl cellulose), or a chitosan-based gel (e.g., chitosanglycerophosphate). Additional, non-limiting examples of drug-elutingpolymers that can be used to formulate any of the pharmaceuticalcompositions described herein include, carrageenan,carboxymethylcellulose, hydroxypropylcellulose, dextran in combinationwith polyvinyl alcohol, dextran in combination with polyacrylic acid,polygalacturonic acid, galacturonic polysaccharide, polysalactic acid,polyglycolic acid, tamarind gum, xanthum gum, cellulose gum, guar gum(carboxymethyl guar), pectin, polyacrylic acid, polymethacrylic acid,N-isopropylpolyacrylomide, polyoxyethylene, polyoxypropylene, pluronicacid, polylactic acid, cyclodextrin, cycloamylose, resilin,polybutadiene, N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer,maleic anhydrate-alkyl vinyl ether, polydepsipeptide,polyhydroxybutyrate, polycaprolactone, polydioxanone, polyethyleneglycol, polyorganophosphazene, polyortho ester, polyvinylpyrrolidone,polylactic-co-glycolic acid (PLGA), polyanhydrides, polysilamine, polyN-vinyl caprolactam, and gellan.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutic amount is one that achievesthe desired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a therapeutic compound (i.e., aneffective dosage) depends on the therapeutic compounds selected. Thecompositions can be administered one from one or more times per day toone or more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the therapeutic compounds described herein caninclude a single treatment or a series of treatments.

Dosage, toxicity and therapeutic efficacy of the therapeutic compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds which exhibit high therapeutic indicesare preferred. While compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 MDR1/P-Gp is Overexpressed in the Epithelial Layer of SinusMucosa in Subjects Having Chronic Rhinosinusitis with or without NasalPolyposis Relative to Control Subjects and Non-Diseased AdjacentIntranasal Subsites

While the expression of P-gp has been studied in the lower airway, verylittle is known with respect to its presence in the upper airway or itsrelationship to chronic sinonasal inflammation. Furthermore, there is noreport on the expression of P-gp across disease states or between sinusand adjacent intranasal sub sites. Therefore, the pattern and degree ofepithelial P-gp expression was examined in three subject groups: thecontrol subjects; the subjects having chronic sinusitis (CRS) withoutnasal polyposis; and the subjects having chronic sinusitis with nasalpolyposis (CRSwNP).

Control subjects were defined as subjects free of rhinosinusitis.Subjects were defined as having CRS with or without NP using theestablished consensus diagnostic criteria (Rosenfeld et al., OtolaryngolHead Neck Surg. 2007 September; 137(3 Suppl):S1-31). Exclusion criteriaincluded the following: use of steroids or immunotherapy within thepreceding 4 weeks, aspirin sensitivity (ASA triad), ciliary dysfunction,autoimmune disease, cystic fibrosis or any known immunodeficiency.Tissues were collected from the septum, inferior turbinate, andparanasal sinuses (or polyp in CRSwNP) in each subject.

Following mucosal sampling, the tissues were immediately snap frozen inOCT. Four samples per subsite were sectioned at 10 μm and preserved in4° C. acetone. Following blocking, the primary antibody (monoclonalanti-P-gp clone F4, 1:250, Sigma Aldrich, St. Louis, Mo.) was appliedfor 24 h at 4° C. The tissue was then rinsed followed by application ofthe secondary antibody (Anti-Mouse IgG (Fc specific) F(ab′)2fragment-FITC, 1:160, Sigma Aldrich, St. Louis, Mo.) for 30 minutes atroom temperature. Slides were then rinsed and mounted in Vectashieldcontaining propidium iodide (Vector Laboratories, Burlingame, Calif.)for nuclear counterstaining. Negative control slides were consideredthose in which the primary antibody was omitted from the stainingprocedure.

Quantitative fluorescent immunohistochemistry (Q-FIHC) for membranousP-gp expression was performed using previously described techniques(Kirkeby et al., J Immunol. Methods. 2005 June; 301(1-2):102-13).Briefly, standardized image capture was performed with an uprightepifluorescent microscope (Carl Zeiss, Oberkochen, Germany) using theFITC filter following a standard 1000 ms exposure. Imaging sites werechosen based on morphology using the nuclear stain to eliminate thepotential for bias. Images were then exported in tagged image fileformat (TIFF) into a graphics editing program (Adobe Photoshop v8.0, SanJose, Calif.). Images were cropped to exclude all non-tissue bearingregions. The lasso tool was used to partition the image into epithelialand stromal regions. The partitioning accuracy was verified usingmatched propidium iodide nuclear stained images. Following partitioning,the magic wand tool (tolerance 30) was used to select pixels exceedingthe luminosity tolerance threshold in each compartment. Fluorescence ina relatively acellular region of the stromal compartment was considerednon-specific background staining and used as an internal control foreach slide. An average luminosity score reflecting the epithelial andstromal compartments was generated and an epithelial/stromal luminosityratio was recorded for each slide.

For statistical analysis, the sample size of four subjects per subgroupwas determined by a power analysis assuming a 1-β of 80% and asignificance level of p<0.05. Staining ratios of 1 or less wereconsidered negligible expression above background. The significance ofdifferences between P-gp expression ratios was determined using a 2tailed Student's t-test (SigmaStat v3.1, Systat Software Inc, San Jose,Calif.).

Among the sinus mucosal specimens, P-gp expression in the CRSwNP group(n=4, 1.570+/−0.354) was significantly greater than both the CRS group(n=4, 1.224+/−0.248) and the control group (n=4, 0.762+/−0.128)(p<0.001, p=0.002; respectively) (FIG. 1, 2). P-gp expression in the CRSgroup was significantly greater than the control group (p=0.002) (FIG.1, 2). Among the negative control slides, there was no differencebetween the staining ratios of the CRSwNP, CRS, or control samples(0.889+/−0.125, 0.982+/−0.030, and 0.929+/−0.137, n=4, respectively).

Among the septal mucosal samples, there was no significant differencebetween CRSwNP (n=4, 0.914+/−0.264), CRS (n=4, 1.126+/−0.476), orcontrol (0.966+/−0.327) tissues (FIG. 1). Among the inferior turbinatemucosal samples, there was no significant difference between CRSwNP(n=4, 1.047+/−0.157), CRS (n=4, 1.099+/−0.362), or control tissues (n=4,0.824+/−0.181).

When examined under high magnification, P-gp staining in the CRSwNP andCRS samples was localized to both the apical and basolateral aspects ofthe epithelial cell membranes (FIG. 3).

These results show that membrane-bound P-gp protein expression wasupregulated in the sinus epithelial cells in the subjects having CRSwNPor CRS relative to the control subjects. Within the same subjects,expression in adjacent non-diseased subsites was not similarlysignificantly elevated. Thus P-gp may play a role in theetiopathogenesis of rhinosinusitis.

Example 2 Membranous P-Glycoprotein is Expressed in the Primary NasalEpithelial Cells

The primary nasal epithelial cell cultures (PNECCs) were generated fromthe sinus mucosal biopsies from five subjects who are free ofrhinosinusitis and undergoing surgery for either cerebrospinal fluidleak repair or tumor removal. Exclusion criteria included the following:diagnosis of chronic sinusitis, use of oral steroids or immunotherapywithin the preceding 4 weeks, aspirin sensitivity, ciliary dysfunction,autoimmune disease, cystic fibrosis, or any known immunodeficiency. Alltissues were derived from schneiderian mucosa within the middle meatus.

Mucosal biopsies were washed and digested in Pronase for 90 minutes at37° C. Cell suspensions were separated from particulate matter bycentrifugation and resuspended in basal epithelial growth medium (BEGM)(Lonza, Basel, Switzerland). Cells were plated for 2 hours on standardtissue culture plates to remove contaminating fibroblasts. Cells werethen expanded for 3-5 days on collagen coated 75 cm² dishes (CorningLife Sciences, Corning, N.Y.). Once confluent, the PNECCs weretrypsinized and re-seeded evenly on human collagen type IV-coated 6-welltissue culture plates. Cultures were grown to 80% confluence in BEGMprior to testing. PNECC cells intended for immunohistochemistry weregrown on tissue culture treated coverslips placed within the wells.

PNECC cells were exposed to 23 hours of stimulation withlipopolysaccharide (LPS, 0.05 mg/mL), which is a toll-like receptor 4agonist and capable of eliciting a strong immune responses. Controlcells were considered those exposed to culture medium alone (BEGM). A0.4% trypan blue (Sigma, St. Louis, Mo.) cell survival assay was used toensure the stimulant exposures were not cytotoxic. In all wells, lessthan 20% of cells were stained blue indicating greater than 80% survivalof the cells. Following the LPS exposures, the media was removed fromeach well.

Q-FIHC for membranous P-gp expression was performed using previouslydescribed techniques (Kirkeby et al., 2005). Briefly, PNECC cells werefixed in 4° C. acetone. Following blocking, the primary antibody(monoclonal anti-P-gp clone F4, 1:250, Sigma Aldrich, St. Louis, Mo.)was applied for 24 h at 4° C. The cells were then rinsed followed byapplication of the secondary antibody (Anti-Mouse IgG (Fc specific)F(ab′)2 fragment-FITC, 1:160, Sigma Aldrich, St. Louis, Mo.) for 30 minat room temperature. The coverslips were then rinsed and mounted inVectashield containing propidium iodide (Vector Laboratories,Burlingame, Calif.) for nuclear counterstaining. Negative control slideswere considered those in which the primary antibody was omitted from thestaining procedure. The mean corrected luminosity was considered thestaining intensity (calculated using Image J v1.45s) divided by thetotal number of pixels subtended by the cells.

For statistical analysis, the sample size of five subjects wasdetermined by a power analysis assuming a 1-β of 80% and a significancelevel of p<0.05. The significance of differences in membranous P-gpexpression was determined using 2 tailed Student's t-tests with post-hoctesting using the Bonferroni procedure (SigmaStat v4, Systat SoftwareInc, San Jose, Calif.).

Membranous P-gp was detected by Q-FIHC in submerged PNECCs grown inculture media alone (BEGM) with a mean corrected luminosity of 1.27 (95%CI 0.41 to 2.13). Exposure of PNECCs to 23 h of LPS resulted in asignificant increase in P-gp expression with a mean corrected luminosityof 9.64 (95% CI 4.30 to 14.98, p=0.017) (FIGS. 4 and 5).

This increase in P-gp expression was confirmed by ELISA followingmembrane extraction. Following removal of media from each well, thecytoplasmic and membranous protein fractions were isolated using atwo-step extraction assay (DualXtract, Bulldog Bio, Inc., Portsmouth,N.H.). Membrane bound P-gp was quantified by subjecting the membranousfraction to ELISA (USCN Life Sciences Inc., Wuhan, P.R. China) andnormalized to the cytoplasmic protein concentration. Normalizedmembranous P-gp was significantly greater in LPS-exposed PNECCs than incontrol cells (mean, 95% CI; 19.63, 11.62 to 27.64 vs. 4.42, 2.88 to5.97, respectively, p=0.016) (FIG. 6).

The immunohistochemical and ELISA data show that P-gp was both presentand functional within PNECC. The primary antibody used in our Q-FIHCstudy is specific to an extracellular loop of the P-gp protein providingfurther confirmation that our data reflects the activity of the membranebound P-gp as opposed to the cytoplasmic fraction which does notparticipate in substrate transport out of the cell.

In some wells, a specific P-gp inhibitor (PSC 833 8micM, TocrisBioscience, Bristol, UK) was applied to the cells 1 hour prior to LPSstimulation. Following the LPS and PSC 833 treatment, the media wasremoved from each well, and ELISA was performed to quantify membranebound P-gp. LPS stimulated membranous P-gp did not change significantlywhen an 8micM PSC 833 solution was added as compared to LPS alone(70.40, 22.77 to 118.04; p=0.115) (FIG. 6).

Example 3 P-Glycoprotein is Responsive to Selective Inhibition In Vitro

The primary nasal epithelial cell cultures (PNECCs) were generated aspreviously described in Example 2. Specific P-gp inhibitors (PSC 8338micM or 80micM, Tocris Bioscience, Bristol, UK; and verapamil 10micM or100micM, Sigma, St. Louis, Mo.) were added to the culture medium for 21hours. Verapamil is a first-generation P-gp inhibitor. PSC 833 is a“second-generation” P-gp specific inhibitor that lacks immunosuppressiveactivity. PSC 833 is thought to impair both the ATPase activity as wellas the transport function of P-gp as a high affinity competitivesubstrate (Morjani et al., Methods Mol. Biol. 2010; 596:433-46).

Rhodamine 123 accumulation assay was then performed. Rhodamine 123(500micM, Sigma, St. Louis, Mo.), a P-gp specific substrate, was thenadded to each well for 2 hours. The rhodamine 123 was then removed andthe P-gp inhibitor alone was added back to the wells for 1 hour. Themedia was then removed and the cells were lysed using Cell Lytic M(Sigma, St. Louis, Mo.). The total intracellular rhodamine 123concentration in each well was determined by spectrophotometry(excitation 510 nm, emission 534 nm) and normalized to the totalcytoplasmic protein using a Pierce BCA Protein Assay Kit (ThermoScientific, Waltham, Mass.). Retention of intracellular rhodamine 123over baseline was considered proportional to degree of P-gp inhibition.

The Rhodamine 123 accumulation assay shows that selective inhibition ofP-gp led to a significant increase in intracellular rhodamine 123 overbaseline in a dose-dependent fashion. Exposure to an 8micM solution ofPSC 833 resulted in a mean 118.12+/−12.16% increase in accumulated P-gpwhile an 80micM demonstrated a significant mean increase of439.46+/−117.59% (p=0.015) (FIG. 7). A similar dose-dependentaccumulation was seen using a 10micM and 100micM verapamil solution(mean+/−SD, 185.33+/−6.59% vs. 328.27+/−66.98%, respectively, p=0.014)(FIG. 7).

The accumulation assay results show that the low dose PSC 833 utilizedin this study was sufficient to inhibit P-gp mediated transport asevidenced by increased retention of intracellular rhodamine 123 overbaseline. While the dose response seen suggests that P-gp is not fullyinhibited at 8micM of PSC 833, the lower dose was utilized in thefollowing cytokine secretion experiments to prevent the possibility ofcytotoxicity.

Example 4 P-Glycoprotein Participates in Regulation of StimulatedEpithelial Cytokine Secretion In Vitro

The primary nasal epithelial cell cultures (PNECCs) were generated aspreviously described in Example 2. Cells were exposed to 23 hours ofstimulation with LPS (0.05 mg/mL) with or without concomitant P-gpinhibition by PSC 833(8micM), which was applied to cells 1 hour prior toLPS treatment. Control wells were considered those exposed to culturemedium alone (BEGM). A 0.4% trypan blue (Sigma, St. Louis, Mo.) cellsurvival assay was used to ensure the stimulant and inhibitor exposureswere not cytotoxic. In all wells less than 20% of cells were stainedblue indicating greater than 80% survival. Following the LPS and PSC 833exposures, the media was removed from each well. Cytokine concentrationsfor IL-6, IL-8, GM-CSF, and TSLP in each well were determined by ELISAaccording to the manufacturer guidelines (eBioscience, San Diego,Calif.). Cytokine concentrations were normalized to total media proteinconcentrations using a Pierce BCA Protein Assay Kit.

With respect to normalized IL-6, PNECCs demonstrated a detectablebaseline secretion that was non-significantly upregulated following LPSstimulation (mean, 95% CI; 57.95, 30.58 to 85.37 vs. 79.67, 42.26 to117.07, respectively, p=0.082). Stimulated IL-6 secretion wassignificantly decreased following P-gp inhibition (mean 37.60, 95% CI11.54 to 63.65, p=0.023) (FIG. 8).

With respect to normalized GM-CSF, PNECCs demonstrated a detectablebaseline secretion that was significantly upregulated following LPSstimulation (mean, 95% CI; 4.45, −0.88 to 9.77 vs. 39.92, 7.90 to 71.94,respectively, p=0.049). Stimulated GM-CSF secretion was significantlydecreased following P-gp inhibition (mean 7.64, 95% CI 2.25 to 13.03,p=0.044) (FIG. 9).

With respect to normalized TSLP, PNECCs demonstrated a detectablebaseline secretion that was non-significantly upregulated following LPSstimulation (mean, 95% CI; 7.27, 6.23 to 8.30 vs. 6.65, 5.35 to 7.96,respectively, p=0.2′74). Stimulated TSLP secretion was significantlydecreased following P-gp inhibition (mean 5.13, 95% CI 4.44 to 5.82,p=0.038) (FIG. 10).

With respect to normalized IL-8, PNECCs demonstrated a detectablebaseline secretion that was significantly upregulated following LPSstimulation (mean, 95% CI; 263.81, 157.22 to 370.40 vs. 912.91, 466.891358.92, respectively, p=0.018). Stimulated IL-8 secretion demonstrateda trend towards reduction following P-gp inhibition although this wasnot significant (mean 801.09, 95% CI 596.88 to 1005.30, p=0.313) (FIG.11).

Among the cytokines sensitive to PSC 833 exposure, the LPS stimulatedsecretion following P-gp inhibition was equivalent to or significantlyless than baseline levels (IL-6, p=0.014; GM-CSF, p=0.001; TSLP,p=0.008).

The cytokine assays demonstrated that inhibition of P-gp resulted in asignificant reduction in LPS stimulated IL-6, GM-CSF, and TSLPsecretion. The lack of significant inhibition of IL-8 suggests that P-gpmediated immunomodulation is selective and does not apply to allsecreted cytokines. The stable P-gp expression in LPS stimulated cellsexposed to PSC 833 as compared to LPS alone suggests that the reductioneffect cannot be attributed to a down-regulation in epithelial P-gp. Therhodamine 123 accumulation assay confirms that PSC 833 is mediating itseffect through P-gp specific inhibition.

As demonstrated herein, P-glycoprotein is overexpressed in sinonasalinflammation. In addition, P-gp functions as an immunomodulator throughregulation of epithelial cytokine secretion. P-gp therefore contributesto the etiopathogenesis of sinonasal inflammation.

Example 5 P-Glycoprotein Inhibition in Epithelial Cells Results inIncreased Intracellular Steroid Retention and Potentiates theAnti-Inflammatory Effect of Steroid

Primary sinonasal epithelial cells were obtained from three patientshaving chronic rhinosinusitis with nasal polyps, who were treated withsteroid dexamethasone. These cultured primary sinonasal epithelial cellswere stimulated with lipopolysaccharide (LPS) in the presence or absenceof the P-gp inhibitor PSC 833. Treatment with PSC 833 resulted inincreased intracellular dexamethasone retention relative to the cellstreated with dexamethasone alone in all three patients (FIG. 12). Themean increase in the normalized dexamethasone retention wasstatistically significant by t-test (FIG. 12). Several steroids areknown substrates of P-glycoprotein; a reduction in P-gp-mediated effluxresults in increased retention of steroid, which may potentiate orsensitize the cells to the therapeutic effects of steroid.

The steroid retention effect was confirmed in nasal polyp explants(n=12/group) treated with two different corticosteroid (dexamethasone orprednisone) and two independent P-gp inhibitors (Verapamil orZosuquidar). FIG. 13 illustrates the normalized intracellularcorticosteroid concentration of nasal polyp explants after exposure toeither dexamethasone (0.05 mg/mL) or prednisone (0.05 mg/mL) in thepresence of media alone or the P-gp inhibitor Verapamil (12.5 μM) orZosuquidar (0.31 μM). A statistically significant increase inintracellular prednisone retention was observed in the nasal polypexplants after exposure to either Verapamil or Zosuquidar (FIG. 13A). Astatistically significant increase in intracellular dexamethasoneretention was observed after Verapamil exposure (FIG. 13B). A similartrend of increase in intracellular dexamethasone retention was seenafter Zosuquidar exposure (FIG. 13B).

The ability of nasal polyp explants to retain steroid after a 30-minuteincubation in dexamethasone (0.05 mg/mL, n=6) was examined. Followingincubation, polyp explants were placed either in media alone or in mediacontaining the P-gp inhibitor PSC 833 (8 μM) and the amount of steroidreleased by the explants into the surrounding media was measured. Astatistically significant decrease in steroid release was observed inexplants exposed to PSC 833, relative to explants exposed to media alone(FIG. 14). These data indicate that blocking P-gp pump action preventsdexamethasone from being cleared by the cells and thereby enhances itstherapeutic efficacy.

The increase in intracellular corticosteroid concentration potentiatesits anti-inflammatory effect. Primary sinonasal epithelial cell culturewas derived from patients with chronic sinusitis with nasal polyps(CRSwNP) and stimulated with LPS, a component of gram negative bacterialcell walls. FIG. 15 shows that the control cells that were notstimulated with LPS had very low baseline level of GM-CSF secretion, LPS(0.0125 mg/ml) treatment increased GM-CSF secretion. Treatment with P-gpinhibitor PSC833 (80 μM) and dexamethasone (0.1 mg/mL) reduced GM-CSFsecretion in cells stimulated with LPS when compared to dexamethasonetreatment alone (FIG. 14).

Example 6 P-Glycoprotein Promotes Epithelial Th2 Associated CytokineSecretion in Chronic Sinusitis with Nasal Polyps

Chronic sinusitis with nasal polyps (CRSwNP) is characterized by thepresence edematous polypoid mucosa and eosinophilic inflammation (ChinD, Harvey R J., Curr. Opin. Otolaryngol. Head Neck Surg. 21: 23-30,2013). While multiple etiologic hypotheses have been explored, recentevidence has focused on the sinonasal epithelial cell as a primarydriver of the local dysregulated immune response through secretion ofT-helper (Th)2 promoting cytokines (Sachse F, Becker K, von Eiff C,Allergy 65:1430-7, 2010; Damm M, Quante G, Rosenbohm J, Otolaryngol HeadNeck Surg. 134:245-9, 2006). Although a role of P-gp in theetiopathogensis of CRSwNP has been suggested, its specific capability ofmodulating Th2 associated cytokines in nasal polyps has not beenexplored.

The sinonasal epithelium functions as a barrier organ against theexternal environment and is endowed with an array of innate and adaptiveimmunologic mechanisms to combat extrinsic pathogens (Lane A P,Truong-Tran Q A, Schleimer R P. Am J Rhinol. 20:138-44, 2006; Bachert C,Gevaert P, van Cauwenberge P., Allergy 57:480-7, 2002). While theselocal exposures may lead to mucosal inflammation, among patients withnasal polyps, the persistence of eosinophilic disease in the face ofantimicrobial therapy suggests an alternate mechanism. Recent studieshave demonstrated that the epithelial cell is capable of independentlypromoting Th2 inflammation in CRSwNP (Van Crombruggen K, Zhang N,Gevaert P, et al., J Allergy Clin Immunol 128:728-32, 2011). Olze et al.demonstrated that the eosinophil chemoattractants, Eotaxins 1, 2, and 3,are upregulated in nasal polyp tissue (Olze H, Förster U, Zuberbier T,Rhinology 44:145-50, 2006). Subsequent in vitro studies have localizedeotaxin to epithelial cells and have demonstrated that both eotaxin 3and acidic mammalian chitinase (AMCase), another pro-Th2 mediator, areupregulated in response to exposure to chitin (Lalaker A, Nkrumah L, LeeW K, et al. Am J Rhinol Allergy 23:8-14, 2009). Exposure toStaphylococcus aureus has also been shown to induce IL-6 production innasal polyp derived epithelial cells. This is postulated to promote Th2activity by counteracting IL-10 induced regulatory T-cell suppression(Sachse F, Becker K, von Eiff C, Allergy 65:1430-7, 2010; Damm M, QuanteG, Rosenbohm J, Otolaryngol Head Neck Surg. 134:245-9, 2006).

While these studies suggest that epithelial cells are capable oforchestrating a local immune response, the post-translational mechanismsgoverning cytokine secretion are less understood. The classic orcanonical pathway involves cytosolic translocation of cytokineprecursors to the endoplasmic reticulum (ER) under the direction of asignal peptide. Non-canonical pathways have also been described whichmay confer greater selective control over cytokine release into thetissue microenvironment (Nickel W., Eur. J Biochem. 270:2109-19, 2003).

P-gp mediated cytokine regulation represents an establishednon-canonical pathway which has been reported in a variety of tissuesincluding, T-cells (Kooij G, Backer R, Koning J J, PLoS One 4:e8212,2009), Schwann cells (Marty V, Medina C, Combe C, Glia 49:511-9, 2005),and the HCT16 and HCT116 colon carcinoma cell lines (Stein U, Walther W,Shoemaker R H., Br J Cancer 74:1384-91, 1996). The previous examplesdemonstrate that a significant reduction in several Th2-associatedcytokines following selective P-gp inhibition in lipopolysaccharide(LPS)-stimulated epithelial cultures derived from healthy mucosa; andthat the epithelial P-gp was overexpressed in nasal polyps relative tocontrol tissue. Taken together, these findings suggest a potential rolefor P-gp in the etiopathogenesis of CRSwNP. The capability of P-gp inmodulating epithelial derived Th2 associated cytokine secretion in nasalpolyps was therefore explored.

Sinus mucosal biopsies were procured from subjects who met the 2012EPOS16 criteria for chronic sinusitis with nasal polyps. Exclusioncriteria included the following: use of topical/oral steroids orimmunotherapy within the preceding 4 weeks, aspirin sensitivity, ciliarydysfunction, autoimmune disease, cystic fibrosis, or any knownimmunodeficiency.

Immediately following harvest of nasal polyp and septal mucosal samplesin 4 patients, tissue explants were cut into 3×3 mm cubes and incubatedfor 30 min at 37° C. in basal epithelial growth medium (BEGM) (Lonza,Basel, Switzerland) with or without 8 μM of the P-gp inhibitor PSC 833(Tocris Bioscience, Bristol, UK). Calcein Acetoxymethylester (AM) 2.5 μM(BD Biosciences, Franklin Lakes, N.J.), a P-gp specific substrate, wasthen added to each sample and incubated for an additional 15 min at 37°C. All tissue was then snap frozen, sectioned, and imaged usingexcitation and emission filters of 467-498 nm and 513-556 nm,respectively for 500 ms. The epithelial and background luminosity wascalculated using Image J (v1.45s). A corrected epithelial luminosity wascalculated by dividing the epithelial and background values to normalizeany variability in sample preparation or image capture.

Human sinonasal nasal epithelial cell cultures (HSNECCs) from fivepatients were grown from nasal polyps as previously described. Briefly,polyp samples were washed and digested in Pronase for 90 minutes at 37°C. Cell suspensions were separated from particulate matter bycentrifugation and resuspended in BEGM. Cells were plated for 2 hours onstandard tissue culture plates to remove contaminating fibroblasts.Cells were then expanded for 3-5 days on collagen coated 75 cm² dishes(Corning Life Sciences, Corning, N.Y.). Once confluent, the HSNECCs weretrypsinized and re-seeded evenly on human collagen type IV-coated 6-welland 96-well tissue culture plates. 6-well cultures were grown to 80%confluence in BEGM prior to cytokine and protein analysis. 96-wellplates intended for the dose dependent inhibition assay were grown to100% confluence. Cells intended for immunohistochemistry were grown ontissue culture treated coverslips.

Membrane-bound P-glycoprotein in HSNECCs was quantified as follows.After removal of media from each well, the cytoplasmic and membranousprotein fractions were isolated using a two-step extraction assay(DualXtract, Bulldog Bio, Inc., Portsmouth, N.H.). Membrane-bound P-gpwas quantified by subjecting the membranous fraction to ELISA (USCN LifeSciences Inc., Wuhan, P.R. China) and normalized to the totalcytoplasmic protein concentration using a Pierce BCA Protein Assay Kit(Thermo Scientific, Waltham, Mass.).

Fluorescent immunohistochemistry (FIHC) for membranous P-gp expressionwas performed as described above. Briefly cells were fixed in 4° C.acetone. Following blocking, the primary antibody (monoclonalanti-P-glycoprotein clone F4, 1:250, Sigma Aldrich, St. Louis, Mo.) wasapplied for 24 h at 4° C. The tissue was then rinsed followed byapplication of the secondary antibody (Anti-Mouse IgG (Fc specific)F(ab′)2 fragment-FITC, 1:160, Sigma Aldrich, St. Louis, Mo.) for 30minutes at room temperature. The coverslips were then rinsed and mountedin Vectashield containing propidium iodide (Vector Laboratories,Burlingame, Calif.) for nuclear counterstaining. Negative control slideswere considered those in which the primary antibody was omitted from thestaining procedure.

PSC 833 dose-dependent inhibition assay was performed as follows. PSC833 was added to confluent HSNECC cultures in a 96-well plate atconcentrations ranging from 0-1.25 μM. Following incubation for 30minutes at 37° C., 2.5 μM of calcein AM was added for an additional 15minutes. Cells were then washed with cold PBS and the totalintracellular calcein concentration in each well was determined byfluorescent spectrophotometry (excitation 494 nm, emission 517 nm).Retention of intracellular calcein relative to the uninhibited controlwells was considered proportional to P-gp inhibition.

The sample size was determined by a power analysis assuming a 1-βof 80%and a significance level of p<0.05. The significance of differencesbetween P-gp activity in mucosal explants, normalized cytokinesecretion, and membranous P-gp expression were determined using anon-parametric, two-tailed, Mann-Whitney U test. The correlation betweenLPS stimulated cytokine secretion and membranous P-gp expression wasdetermined using a Pearson product-moment correlation coefficient. Thedifferences P-gp inhibition using PSC 833 were determined using a onetailed Mann-Whitney U test.

Following incubation of both nasal polyp and septal explants (n=4, each)in media containing calcein AM (2.5 μM), no significant difference inintraepithelial calcein retention as measured by epithelial correctedluminosity was found between sites (mean+/−SD; 2.55+/−0.62 vs.2.32+/−0.86; respectively; p=NS). Among the septal explants, nosignificant change in calcein retention was seen following P-gpinhibition with 8 μM PSC 833 (2.22+/−0.92). Among the nasal polypsexplants, P-gp inhibition resulted in a significant increase inintraepithelial calcein retention as compared to the uninhibited samples(5.17+/−1.76; p<0.05) (FIG. 16). Thus epithelial P-gp is hyperfunctionalin nasal polyps as compared to adjacent non-polypoid mucosa.

These findings demonstrated a rise in intraepithelial calcein in polypas compared to septal explants following inhibition with a P-gp specificinhibitor PSC 833. This suggests that P-gp activity in polyps is focallyincreased relative to adjacent healthy mucosa (Morjani H, Madoulet C.Methods Mol Biol 596:433-46, 2010; Iqbal M, Gibb W, Matthews S G.Endocrinology 152:1067-79, 2011). Example 1 showed that the presence ofmembranous P-gp overexpression in CRSwNP as compared to non-diseasedsubsites. This assay therefore provides supporting evidence suggestingthat this overexpression is also associated increased pump activity innasal polyps.

P-gp was detected in the membrane extract of all CRSwNP HSNECCs by ELISA(n=5). The mean normalized concentration in cells exposed to LPS 0.05mg/mL (22.23+/−10.22) was significantly greater than in cells exposed tomedia alone (5.91+/−3.15, p<0.05). The presence of membranous P-gpexpression was confirmed by FIHC using a primary antibody targeted tothe extracellular loop of P-gp (FIG. 17).

Calcein fluorescence in confluent HSNECCs (n=5) increased in adose-dependent manner following exposure to successively higherconcentrations of PSC 833 (FIG. 18). The percent fluorescence relativeto uninhibited control wells at 1.25 μM PSC 833 (134.32+/−43.78) wassignificantly greater than at 0.31 μM (89.07+/−28.56; p=0.04) but not at0.63 μM (102.58+/−44.32; p=NS) (FIG. 18).

For P-gp mediated cytokine secretion testing, HSNECC cells were exposedto 0.05 mg/mL LPS stimulation for 23 hours with or without concomitantP-gp inhibition using PSC 833 (8 μM), which was applied one hour priorto LPS stimulation. Control wells were considered those exposed toculture medium alone (BEGM). A 0.4% trypan blue (Sigma, St. Louis, Mo.)cell survival assay was used to ensure the stimulant and inhibitorexposures were not cytotoxic. In all wells less than 20% of cells werestained blue indicating greater than 80% survival. Following the LPS andPSC 833 exposures, the media was removed from each well. Cytokineconcentrations for GM-CSF, IL-6, IL-8, and IL-25 in each well weredetermined by ELISA according to the manufacturer guidelines (GM-CSF,IL-6, IL-8 eBioscience, San Diego, Calif.; IL-25 Assay Biotechnology,San Francisco, Calif.). Cytokine concentrations were normalized to totalmedia protein concentrations using a Pierce BCA Protein Assay Kit.

The basal secretion of GM-CSF (pcg/mL) normalized to total media protein(mcg/mL) was 4.33+/−1.12. Exposure to LPS 0.05 mg/mL resulted in asignificant increase in secretion (45.21+/−41.39; p<0.01) which wassignificantly inhibited by the addition of PSC 833 8 μM (8.47+/−3.28;p<0.01). The concentration of LPS stimulated GM-CSF secretion was highlycorrelated with the degree of membranous P-gp expression (r=0.824,p<0.05) (FIG. 19).

The basal secretion of IL-6 (pcg/mL) normalized to total media protein(mcg/mL) was 71.15+/−44.93. Exposure to LPS 0.05 mg/mL did notsignificantly alter IL-6 secretion (63.16+/−36.37, p=NS). The additionof PSC 833 8 μM significantly inhibited LPS stimulated IL-6 secretion(39.94+/−31.07; p<0.05). The concentration of LPS stimulated IL-6secretion was highly correlated with the degree of membranous P-gpexpression (r=0.833, p<0.05) (FIG. 19).

The basal secretion of IL-8 (pcg/mL) normalized to total media protein(mcg/mL) was 316.47+/−174.42. Exposure to LPS 0.05 mg/mL resulted in asignificant increase in secretion (1041.17+/−550.52; p<0.05). Noinhibition of IL-8 secretion was seen following the addition of PSC 8338 μM (1608.41+/−471.94). The concentration of LPS stimulated IL-8secretion did not correlate with the degree of membranous P-gpexpression (r=−0.011, p=NS) (FIG. 20).

The basal secretion of IL-25 (pcg/mL) normalized to total media protein(mcg/mL) was 0.031+/−0.019. Exposure to LPS 0.05 mg/mL resulted in asignificant increase in secretion (0.075+/−0.039; p<0.05). No inhibitionof IL-25 secretion was seen following the addition of PSC 833 8 μM(0.073+/−0.038; p=NS). The concentration of LPS-stimulated IL-25secretion did not correlate with the degree of membranous P-gpexpression (r=0.008, p=NS) (FIG. 20).

The majority of chronic sinusitis with nasal polyps is associated witheosinophilic infiltration and a predominantly Th2 cytokine profile(Mjosberg J M, Trifari S, Crellin N K, Nat. Immunol. 12:1055-62, 2011).Multiple lines of evidence suggest that the respiratory epithelial cellis capable of elaborating a variety of cytokines such as IL-6, IL-25,IL-33, TSLP, and GM-CSF which can not only activate Th2 cells (Peters AT, Kato A, Zhang N, J Allergy Clin Immunol 125:397-403, 2010; Reh D D,Wang Y, Ramanathan M Jr, Am J Rhinol Allergy 24:105-9, 2010; WisniewskiJ A, Borish L. Allergy Asthma Proc 32:83-94, 2011), but may alsostimulate the recently described type 2 innate lymphoid cell (ILC) toproduce T-cell independent Th2 polarizing cytokines (Mjösberg J M,Trifari S, Crellin N K, Nat. Immunol. 12:1055-62, 2011). Despite thesefindings, a complete understanding of the mechanisms responsible formaintaining the chronic inflammation seen in CRSwNP remains elusive.Non-canonical regulation of epithelial cytokine secretion via P-gp mayrepresent one such mechanism.

Primary sinonasal epithelial cell culture model derived from nasalpolyps was utilized to further explore the immunomodulatory role ofP-gp. The FIHC and ELISA findings confirmed that membranous P-gpexpression both persisted in vitro and was subject to active regulationfollowing TLR4 stimulation with LPS. The in vitro P-gp inhibition assayalso confirmed that P-gp remained sensitive to PSC 833 in a dosedependent manner and was subject to inhibition at concentrations wellbelow those utilized in the cytokine secretory studies. These resultsvalidate the use of the HSNECC model in examining P-gp functionality andsuggest that any changes seen in cytokine release following PSC 833exposure may be directly attributable to impairment of P-gp pumpactivity.

The findings that IL-6 and GM-CSF secretion are reduced followinginhibition with PSC 833 suggest that their release into the local polypmicroenvironment is subject to P-gp mediated regulation. This is furthersupported by the correlation between P-gp expression and secretion amongthese cytokines. Interestingly, this effect seems to be cytokinespecific as both IL-8 and IL-25 release occurred independently of P-gpinhibition or expression.

The presence of an in vitro correlation between P-gp expression andcytokine secretion coupled with the in vivo findings of P-gpoverexpression and hyperactivity in nasal polyps suggests that P-gp mayplay a central role in promoting and maintaining chronic inflammation inCRSwNP. This is further supported by the fact that both IL-6 and GM-CSFare associated with Th2 inflammation, a key feature of CRSwNP. IL-6 haspreviously been reported to be elevated in CRSwNP, is known to activateTh17 cells, and may contribute to the insufficiency of regulatoryT-cells in nasal polyps (Peters A T, Kato A, Zhang N, Allergy Clin.Immunol. 125:397-403, 2010). Similarly GM-CSF has been shown to becapable of activating eosinophils in CRSwNP leading to enhancedeosinophil chemotaxis and prolonged survival (Shin S H, Lee S H, Jeong HS, Laryngoscope 113:1374-7, 2003).

While the pathogenesis of CRSwNP remains elusive, the epithelial cellhas gained attention as a primary driver of the Th2 inflammation whichcharacterizes the disease. These data suggest that P-gp overexpressionmay promote non-canonical Th2 associated epithelial cytokine secretionin nasal polyps. These findings provide a framework for maintainingchronic inflammation in CRSwNP and offer a potential therapeutic target.In light of this novel role for P-gp as an upper respiratory tractimmunomodulator, future efforts will be directed at elucidating themechanism of P-gp overexpression in the setting of CRSwNP.

Example 7 P-Glycoprotein is a Marker of Tissue Eosinophilia andRadiographic Inflammation in Chronic Rhinosinusitis without Nasal Polyps

Chronic rhinosinusitis (CRS) represents a heterogeneous group ofdiseases with a variety of pathophysiologic mechanisms. A broad divisionbetween chronic sinusitis with and without nasal polyps (CRSwNP andCRSsNP, respectively) serves as a widely accepted distinction secondaryto phenotypic differences evident clinically. The immunologic profilesunderlying these disease states suggest that a broader spectrum existswith a predominantly eosinophilic profile subtending not only patientswith CRSwNP but a subset of those with CRSsNP as well. The presence ofeosinophilic chronic sinusitis (ECRS) is clinically relevant as thesepatients share not only the immunologic profile of those with CRSwNP butalso the propensity for greater symptom severity and worse outcomes.

Eosinophilic chronic rhinosinusitis represents a histologic diagnosisconsisting of mucosal eosinophilia evident in biopsy specimens. Whilethe precise definition of ECRS may be debated, Soler has demonstratedthat a cut point of >10 eosinophils per hpf provided the bestcorrelation with patient outcomes (Soler Z M, Sauer D, Mace J, et al.Otolaryngol Head Neck Surg. 142(1):64-71, 2010). ECRS has also beenshown in multiple studies to correlate with worse symptoms (Sun D I, JooY H, Auo H J, et al., Eur Arch Otorhinolaryngol. 266(7):981-6, 2009;Soler Z M, Sauer D A, Mace J, et al., Otolaryngol Head Neck Surg.141(4):454-61, 2009; Lee T J, Liang C W, Chang P H, et al., Auris NasusLarynx. 36(6):655-60, 2009), lower airway hyperactivity (Amorim M M,Araruna A, Caetano L B, et al., Clin Exp Allergy. 2010; 40(6):867-74;Han D H, Kim S W, Cho S H, et al., Allergy 64(1):118-22, 2009), and poorsurgical outcomes (Soler, 2010). These findings may be understood in thecontext of a shared T-helper 2 (Th2)-skewed immunologic profile aspatients with frank nasal polyps (Takeno S, Hirakawa K, Ishino T.Allergol Int. 59(3):247-56, 2010). A parsimonious interpretation ofthese findings suggests that ECRSsNP and CRSwNP may reflect differentphenotypic manifestations of the same etiologic process.

P-gp has been previously demonstrated to participate in non-canonicalcytokine secretion in T-cells (Drach J, Gsur A, Hamilton G, et al.,Blood. 88(5):1747-54, 1996). The overexpression of P-gp in CRSwNPcoupled with its ability to promote cytokine secretion suggests that itmay play a role in the pathogenesis of the eosinophilic inflammationseen in nasal polyps. Given the relationship between CRSwNP and ECRS,P-gp expression in patients with ECRSsNP was compared to that inpatients with CRSsNP.

Sinus mucosal biopsy samples were harvested from the anterior ethmoidsinus of patients having chronic rhinosinusitis. Exclusion criteriaincluded the following: use of oral steroids or immunotherapy within thepreceding 4 weeks, aspirin sensitivity (ASA triad), ciliary dysfunction,autoimmune disease, cystic fibrosis or any known immunodeficiency.Patient with focal etiologies for sinusitis including mucoceles,odontogenic sources, and fungal balls were similarly excluded from thestudy.

Following mucosal sampling, the tissue was stained as previouslydescribed. Briefly, following blocking, the primary antibody (monoclonalanti-p-glycoprotein clone F4, 1:250, Sigma Aldrich, St. Louis, Mo.) wasapplied for 24 h at 4° C. The tissue was then rinsed followed byapplication of the secondary antibody(Anti-Mouse IgG (Fc specific)F(ab′)2 fragment-FITC, 1:160, Sigma Aldrich, St. Louis, Mo.) for 30 minat room temperature. Slides were then rinsed and mounted in Vectashieldcontaining propidium iodide (PI) for nuclear counterstaining. Negativecontrol slides were considered those in which the primary antibody wasomitted from the staining procedure.

Fluorescent staining intensity was quantified using a modification ofpreviously described methods (Bleier B S., Int Forum Allergy Rhinol.2(2):122-5, 2012). Briefly, image capture was performed with an uprightepifluorescent microscope following a standard 1000 ms exposure. Imageswere then exported into Image J (v1.45s). The nuclear stain was used toselect both the epithelium and a non-tissue bearing background regiongenerating a staining intensity ratio. Samples were excluded if theepithelial staining intensity in the negative control slide exceededthat of the adjacent stroma. An epithelial/background staining ratiogreater than or equal to 3 was defined as high P-gp expression. Thisthreshold or cut point was derived from pilot data demonstrating P-gpexpression ratios of less than 3 in nasal septal mucosa, a region with apreviously described low level of basal P-gp epithelial expression.

For each patient, a representative hematoxylin and eosin slide generatedas part of their routine pathologic analysis at surgery was selected.The number of eosinophils per five 400× high powered fields (hpf) wererecorded by two independent and blinded observers as previouslydescribed (Soler Z M, Sauer D, Mace J, et al. Otolaryngol Head NeckSurg. 142(1):64-71, 2010). The values were averaged to generate a meaneosinophil per hpf score for each patient. Radiographic inflammation wasquantified by a single blinded observer using the Lund-Mackay stagingsystem.

P-gp expression ratios, tissue eosinophilia, and radiographic scoresbetween the patient groups were compared with a two tailed Student'st-test using R (v2.15.2, 2012). P values <0.05 were consideredstatistically significant.

Among the 39 patients included in the study, the epithelial/backgroundratio of the high expression group (mean+/−SD, 4.86+/−1.33, n=7 or17.95%) was significantly greater than that of the low expression group(1.91+/−0.45, n=32 or 82.05%, p<0.001) (FIG. 21A). While there was arelative predominance of females in the high P-gp expression group,there were no significant differences between the two groups withrespect to patient age or race (Table 1).

TABLE 1 Patient demographics Low expression High expression Age (years)47.8 ± 16.7 44.3 ± 14.8 Female (%) 50 85.7 Male (%) 50 14.3 Caucasian(%) 87.5 85.7 Minority (%) 12.5 14.3

Among the high P-gp expression group, all patients demonstrated greaterthan 10 eosinophils/hpf with a mean of 62.38 (range 10.0-240.6). The lowP-gp expression group demonstrated a mean of 5.11 eosinophils/hpf (range0.0-41.4) which was significantly lower than that of the high expressiongroup (p=0.0003) (FIG. 21B).

The mean Lund-Mackay score was significantly greater among the high P-gpexpression group than that of the low P-gp expression group(11.86+/−2.79 vs. 6.84+/−4.19; p=0.005) (FIG. 21C).

FIGS. 22 A and B are fluorescent immunohistochemical images of mucosadepicting representative (A) low epithelial P-gp expression and (B) highepithelial P-gp expression. FIGS. 22 C and D are matched high powered(400×) H&E stromal images depicting the absence (C) and presence (D) ofmucosal eosinophilia (black arrows denote individual eosinophils). FIGS.22 E and F are coronal CT scans demonstrating increased radiographicinflammation in the patient with high P-gp expression (F) relative tothe patient with low P-gp expression (E).

These data demonstrate that among patients with CRSsNP, P-gpoverexpression predicts tissue eosinophilia. While the mechanisticrelationship between epithelial P-gp and eosinophilic inflammationremains unclear, the common finding of upregulation in both CRSwNP andECRS lends support to the idea that P-gp may play an etiopathologicrole. Although the clinical utility of Lund-Mackay score may be debated,one of its strengths is that it provides an objective reflection of thedegree of global inflammation present in the patient. As with CRSwNP,the intraluminal disease burden seen in patients with ECRS tends to bemore severe involving most if not all of the sinuses. Innon-eosinophilic patients, disease may be more isolated to a specificregion leading to a lower overall Lund-Mackay score even if the localinflammation is quite severe. Consequently, we chose to utilize theLund-Mackay score as an additional surrogate marker of inflammationwhich served to support our histologic findings.

In summary, eosinophilic chronic rhinosinusitis shares a similarclinical and histologic profile with CRSwNP, suggesting the two mayrepresent different manifestations of the same underlying process. InExamples 1-6, P-gp was shown to be overexpressed in CRSwNP and iscapable of modulating epithelial cytokine secretion. Here the datashowed that P-gp is similarly overexpressed in ECRS and is associatedwith radiologic evidence of increased inflammation. These findingsfurther strengthen the link between ECRS and CRSwNP and suggest thepotential for an etiopathologic role for P-gp in both diseases.

Example 8 Osteitis is Associated with P-Glycoprotein Overexpression inPatients with Chronic Sinusitis without Nasal Polyps

Chronic rhinosinusitis (CRS) associated with T-helper cell type 2 (Th2)inflammation has been increasingly recognized as a distinct phenotypecharacterized by eosinophilic infiltration. The immunologic profilesunderlying this disease state suggests that eosinophilic chronicrhinosinusitis without nasal polyps (ECRS) and chronic sinusitis withnasal polyps (CRSwNP) may exist along a spectrum of Th2 mediated mucosalinflammation. These patients tend to manifest more severe symptoms andworse outcomes following medical and surgical management. Example 7showed that P-glycoprotein(P-gp) overexpression has been found in bothpatients with ECRS and CRSwNP and is capable of promoting Th2 associatedcytokine secretion. Identification of patients with elevated P-gp maytherefore be useful to guide treatments directed toward this noveltherapeutic target. Radiographic osteitis scores have been previouslyshown to correlate with eosinophilic inflammation and thus may be usefulin stratifying patients with P-gp overexpression (Snidvongs K, McLachlanR, Chin D, et al. Rhinology 2012; 50:299-305; Snidvongs K, McLachlan R,Sacks R, et al. Int Forum Allergy Rhinol 2013; 3:369-375).

Examples 1-6 showed that P-gp functions as an immunomodulator capable ofregulating the efflux of cytokines from its host cell, and suggestedthat P-gp may play a role in the pathogenesis of Th2 mediated sinonasalinflammation. Identification of patients with altered P-gp expressionmay therefore provide prognostic information as well as offer a noveltherapeutic target.

Tissue remodeling is a molecular process of formation and resorptionleading to transient or permanent changes in structure of tissue. InCRS, tissue remodeling is exhibited in both mucosa and bone,characterized by osteitis, mucosal hypertrophy, fibrosis, and thickeningof the basement membrane. Osteitis involves inflammatory changes in theunderlying bone that lead to persistence of disease and is increasinglyrecognized as playing a significant role in recalcitrant CRS (DetwillerK Y, Smith T L, Mace J C, et al. Int Forum Allergy Rhinol 3:364-368,2013). Snidvongs et al. demonstrated that both the Kennedy Osteitis(KOS) and Global Osteitis Scores (GOS) were capable of predictingdisease severity in eosinophilic inflammation (Snidvongs K, McLachlan R,Sacks R, Int Forum Allergy Rhinol 3:369-375, 2013). Thus, whether theseosteitis scores can be used as a non-invasive clinical marker of P-gpoverexpression in patients with CRS was examined.

Sinus mucosal biopsy samples were procured from 38 patients having CRS.CRS was defined using the established consensus diagnostic criteria.Tissue was harvested from the anterior ethmoid sinus of each patient.Exclusion criteria included the following: The presence of nasalpolyposis, use of oral steroids or immunotherapy within the preceding 4weeks, aspirin sensitivity (ASA triad), ciliary dysfunction, autoimmunedisease, cystic fibrosis, or any known immunodeficiency. Patient withfocal etiologies for sinusitis including mucoceles, odontogenic disease,and fungal balls were similarly excluded from the study. Demographicdata was recorded.

Following mucosal sampling, the tissue was stained using quantitativefluorescent immunohistochemistry as previously described. Briefly,following blocking, the primary and secondary antibodies were added for24 h and 30 minutes, respectively followed by the addition of thenuclear counterstain. Negative control slides were considered those inwhich the primary antibody was omitted from the staining procedure.Fluorescent staining intensity was quantified using previously describedmethods. An epithelial/background staining ratio ≥3 was defined as highP-gp expression. This cut point was derived from prior studiesdemonstrating a correlation between staining ratios ≥3 and mucosaleosinophilia in the setting of CRS.

For patient demographics, among the 38 patients included in study, theepithelial/background ratio of the high expression group (mean±SD,4.86±1.33; n=7; 18.42%) was significantly greater than that of the lowexpression group (1.93±0.45; n=31; 81.57%; p<0.001) (FIG. 23A). Whilethere was a relative predominance of females in the high P-gp expressiongroup, there were no significant differences between the two groups withrespect to patient age or race. No patients in the high P-gp expressiongroup had undergone prior surgery while seven (22.6%) patients in thelow P-gp expression group were undergoing revision procedures (Table 2).

TABLE 2 Patient Demographics High P-gp Low P-gp expression groupexpression group Age (years) 40.43 ± 12.07 48.52 ± 16.43 Female (%)51.61 85.71 Male (%) 14.29 48.39 Caucasian (%) 85.71 87.10 Minority (%)14.29 12.9 Prior surgery (%) 0 22.6

Peripheral blood eosinophil counts were available in 25 patients.Eosinophil concentrations were reported as an auto differential result(normal 0-6%) using a Sysmex XS-1000i (Kobe, Japan) instrument. Themethodology of the instrument for the differential is light scatter andfluorescent emission.

Serum eosinophilia was defined as having serum percent eosinophilsgreater than 6%. Patients with high P-gp expression had higher medianserum eosinophil counts (mean±SD, 6.98±2.17) than the low P-gpexpression group (2.36±1.38, p<0.001) (FIG. 23B).

The Kennedy osteitis score (KOS) is determined by using computedtomography (CT) to diagnose osteitis based on the thickness of bonypartitions in the maxillary, ethmoid, and sphenoid sinuses (Lee J T,Kennedy D W, Palmer J N, Am J Rhinol 20:278-282, 2006). Thickness ofbony partitions were measured and classified into the followingcategories of osteitis severity: mild (<3 mm); moderate (4-5 mm); andsevere (>5 mm). This was modified to create a summary score so thatcomparable assessments could be made to the Global osteitis score (GOS)(Georgalas C, Videler W, Freling N, Clin Otolaryngol 35:455-461, 2010).All 10 sinuses (right and left frontal, anterior ethmoid, posteriorethmoid, maxillary, and sphenoid) were scored as being 0 (<3 mm), 1 (3-5mm) or 2 (>5 mm) with total scores ranging from 0 to 20. Woven bone withthickened, irregular, heterogeneous lining of the sinus walls weremeasured rather than normal lamellar/cortical bony wall.

The presence of osteitis was also scored by using the Global osteitisscore (GOS) system proposed by Georgalas (Georgalas C, Videler W,Freling N, Clin Otolaryngol 35:455-461, 2010) and modified by Snidvongs(Snidvongs K, McLachlan R, Sacks R, Int Forum Allergy Rhinol 2013;3:369-375). Osteitis was defined as loss of bone definition,hyperostosis, new bone formation, or signal heterogeneity overlying eachsinus wall. Bony walls of the paranasal sinuses were scored ranging from0 to 4 making the total score of 0 to 40 as previously described (Lee JT, Kennedy D W, Palmer J N, et al. Am J Rhinol 2006; 20:278-282.). AllCT scans were reviewed by a single observer blinded to P-gp expressionand serum eosinophil levels.

Global and Kennedy Osteitis scores were compared using a Pearsoncorrelation coefficient. Correlation(r) values from 0.7-1 wereconsidered strongly correlated. Osteitis scores and serum eosinophilconcentrations between P-gp expression groups were compared using anon-parametric Mann-Whitney U test (two-tailed). P values <0.05 wereconsidered statistically significant.

Both the GOS and KOS were significantly higher among patients with P-gpoverexpression. Among patients with high P-gp expression, the GOS andKOS values (mean±SD, 15.86±4.91 and 6.29±1.25, respectively) weresignificantly greater than those in the low P-gp expression group(4.55±4.33 and 2.23±1.71, p<0.001) (FIG. 24 A, B). When the two scoringsystems were compared to each other, there was a significant correlationbetween KOS and GOS values (r=0.835, p<0.001) (FIG. 24C).

Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of recalcitrantCRS characterized by a T-helper cell type 2 skewing of the localinflammatory milieu. While the etiology of ECRS and Th2 inflammation isunclear, multiple studies have pointed to epithelial cell as a keyparticipant in the inflammatory cascade (Ferguson B J. Curr OpinOtolaryngol Head Neck Surg 12:237-242, 2004; Mehta V, Campeau N G, KitaH, et al. Mayo Clin Proc 83:671-678, 2008). Examples 1-7 demonstratedthat membranous epithelial P-glycoprotein is overexpressed in bothCRSwNP and ECRS and is capable of promoting secretion of Th2 associatedcytokines. These findings suggest that P-gp may play an importantetiopathologic role.

The first large series to report on the association of disease severityand eosinophilia in CRS was published by Newman (Newman L J,Platts-Mills T A, Phillips C D, JAMA 271:363-367, 1994). Multiplesubsequent reports consistently demonstrated that eosinophilia is amarker for more extensive disease that is more refractory to surgicalcure (Zadeh M H, Banthia V, Anand V K, Am J Rhinol 16:313-317, 2002;Szucs E, Ravandi A, Goossens A, Am J Rhinol 16:131-134, 2002). Giventhese findings, a method of identifying these patients preoperativelywould be of considerable value. Osteitis as measured by the two separatescoring systems was shown to correlate with tissue eosinophilia(Snidvongs K, McLachlan R, Sacks R, Int Forum Allergy Rhinol 3:369-375,2013). This study was designed to determine whether osteitis couldsimilarly predict P-gp overexpression.

FIG. 24C confirmed that KOS and GOS are highly correlated and thus bothrepresent an acceptable method of quantifying osteitis. Moreimportantly, FIGS. 24A and 24B showed that a greater osteitis burden isassociated with P-gp overexpression among patients with chronicrhinosinusitis without nasal polyps. FIG. 23 showed that patients withhigh P-gp expression also had higher median serum eosinophil counts thanthe low expression group suggesting that Th2 skewed inflammation may beimplicated as a common link of this relationship.

A potential confounding factor in quantitating bony remodeling is thatosteitis is likely multifactorial and previous studies have suggestedthat surgery itself may play a role in increasing the incidence ofosteitis (Georgalas C. Curr Opin Otolaryngol Head Neck Surg 21:45-49,2013; Cho S H, Shin K S, Lee Y S, Am J Rhinol 22:537-541, 2008;Georgalas C, Videler W, Freling N, Clin Otolaryngol 35:455-461, 2010).However, all patients in the high P-gp expression group were undergoingprimary surgery and thus, at least in this population, surgery can beexcluded as a source of osteitic remodeling.

In summary, P-glycoprotein overexpression in Th2 inflammation representsa recently described phenomenon which may play an important role in theetiopathogenesis of eosinophilic CRS. A method of predicting thispatient population based on objective clinical criteria may be of valueto help to stratify patients for the purposes of tailoring medical andsurgical therapy as well as providing counseling on expectationsfollowing treatment. The data demonstrated that increased osteitisburden among unoperated patients is associated with higher P-gpexpression. The presence of greater serum eosinophilia in the high P-gpexpression group suggest that Th2 inflammation may play a role andfurther studies elaborating the mechanism by which P-gp overexpressionand osteitis are associated are warranted.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of treating chronic rhinosinusitis in asubject, the method comprising: identifying a subject having chronicrhinosinusitis; administering to the subject an effective amount of aP-glycoprotein inhibitor, wherein the P-glycoprotein inhibitor is not anantibiotic agent, an antifungal agent, a corticosteroid, or a firstgeneration P-glycoprotein inhibitor compound.
 2. The method of claim 1,wherein the subject has chronic rhinosinusitis and nasal polyps.
 3. Themethod of claim 1, wherein the P-glycoprotein inhibitor decreasesP-glycoprotein expression in the subject's sinonasal epithelial cells.4. The method of claim 1, wherein the P-glycoprotein inhibitor isadministered systemically.
 5. The method of claim 1, wherein theP-glycoprotein inhibitor is administered locally to the subject's nasalpassage and sinuses.
 6. The method of claim 5, wherein theP-glycoprotein inhibitor is delivered to the subject's nasal passage andsinuses by an inhalation device, by flushing, or by spraying.
 7. Themethod of claim 5, wherein the P-glycoprotein inhibitor is administeredto the subject by a P-glycoprotein inhibitor eluting implant surgicallyplaced in the subject's nasal passage or sinuses.
 8. The method of claim7, wherein the P-glycoprotein inhibitor eluting implant isbioabsorbable.
 9. The method of claim 1, wherein the subject havingchronic rhinosinusitis was identified by endoscopy.
 10. The method ofclaim 1, wherein the subject having chronic rhinosinusitis wasidentified by computed tomography.
 11. The method of claim 1, whereinthe subject having chronic rhinosinusitis was identified by observingthe subject's symptoms and duration of symptoms.
 12. The method of claim1, further comprising monitoring the efficacy of the treatment byendoscopy.
 13. The method of claim 1, further comprising monitoring theefficacy of the treatment by computed tomography.
 14. The method ofclaim 1, further comprising monitoring the efficacy of the treatment byobserving the subject's symptoms and duration of symptoms.
 15. Themethod of claim 1, further comprising surgically removing any nasalpolyps present in the subject.
 16. The method of claim 1, wherein theP-glycoprotein inhibitor is administered in combination with one or bothof a corticosteroid and an antibiotic.
 17. The method of claim 16,wherein the corticosteroid is selected from dexamethasone, prednisone,prednisolone, triamcinolone, cortisol, budesonide, mometasone,fluticasone, flunisolide, and betamethasone.
 18. The method of claim 1,wherein the P-glycoprotein inhibitor is administered in combination witha corticosteroid and an antibiotic.
 19. A kit for treating chronicrhinosinusitis in a subject, said kit comprising a pharmaceuticalcomposition comprising an effective amount of a P-glycoproteininhibitor, wherein the P-glycoprotein inhibitor is not an antibioticagent, an antifungal agent, a corticosteroid, or a first generationP-glycoprotein inhibitor compound; and a device for delivering thepharmaceutical composition to the subject's nasal passage and sinuses,wherein the device delivers the pharmaceutical composition to thesubject's nasal passage and sinuses in a liquid, nebulized, oraerosolized form.
 20. The kit of claim 19, further comprising acorticosteroid.
 21. The kit of claim 19, further comprising anantibiotic.